Antibodies binding to TLR2/TLR4-interacting immunologically active polypeptide

ABSTRACT

Disclosed are immunomodulatory polypeptides that behave as weak TLR2 and TLR4 agonists and as potent competitive antagonists of natural pathogenic ligands for human and murine TLR2 and TLR4, that identify a subset of neutrophils in human peripheral blood leukocytes, and that elicit an unusual induced cytokine profile. Also disclosed are compositions comprising such polypeptides, compositions comprising antibodies that specifically bind to such polypeptides, and methods of using the same, including for treating sepsis or reducing the severity or likelihood of occurrence of sepsis, in cancer treatment, in the treatment of autoimmune diseases, in organ transplantation and for reducing graft rejection, for promoting fertility, and for identifying a neutrophil subset and/or other cellular subset including by flow cytometry. Pharmaceutical compositions and kits, and treatment methods are also disclosed.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 750061_402D2_SEQUENCE_LISTING.txt. The text fileis 112 KB, was created on Jul. 17, 2019, and is being submittedelectronically via EFS-Web.

BACKGROUND Technical Field

Embodiments of the presently disclosed invention relate generally toimmunomodulatory polypeptides, compositions comprising suchpolypeptides, and methods of using the same. More specifically, thepresent embodiments relate to short immunomodulatory polypeptides thatspecifically bind with low affinity to the human and murine toll-likereceptors TLR2 and TLR4 such that they advantageously interfere withTLR2- and/or TLR4-mediated signaling.

Description of the Related Art

Sepsis, Inflammation and Immunity

The interaction between a host organism, such as a human, a mammal oranother vertebrate animal, and a microbial pathogen such as a bacterium,virus, fungus or parasite, is complex and depends on genetic andenvironmental factors (e.g., nutrition, health, temperature); theinterplay of these components may play a defining role in clinicaloutcome. One potential outcome of the host-pathogen interaction may be asubclinical host protective response by which the host eradicates theinfection without any clinical evidence of infection, or, the host andpathogen may engage in a battle that produces clinical symptoms butduring which the host maintains control of the pathogen and eventuallyeradicates it. Alternatively, in sepsis a more intense battle may ensueas a consequence of which the pathogen and/or the host's owninflammatory response eventually overwhelms the host, resulting in deathof the host.

Sepsis is a severe, systemic inflammatory condition that occurs in about750,000 to 900,000 people each year in the United States, andapproximately one-third of sepsis patients die (Kellum et al., 2007 ArchIntern Med 167(15):1655-1663). Published studies have concluded thatsevere sepsis is a common, expensive, and frequently fatal conditionthat deserves more universal attention (Decker, 2004 J. Clin Invest113(10): 1387-1389). Human sepsis may be identified in a patient whenthe patient meets at least two of the four criteria for SystemicInflammatory Response Syndrome (SIRS) and also demonstrates features ofMODS (multiple organ dysfunction syndrome) (Remick, 2007 Am. J. Pathol.170(5):1435-1444). The SIRS criteria include (1) body temperature>38° C.or <36° C., (2) heart rate>90 beats per minute (bpm), (3) respiratoryrate>20 breaths per minute or arterial CO₂<32 mm Hg, and (4) circulatingwhite blood cell (WBC) count>12,000/mm³ or <4000/mm³ or >10% immatureforms (Remick 2007).

The innate immune system depends on the interplay between the pathogenassociated molecular patterns (PAMPs) expressed by bacterial, viral,fungal and parasitic agents, and the pattern recognition receptors(PRRs) expressed by the cells of the host innate immune system,including macrophages, monocytes, neutrophils, and dendritic cells.Association of the immune cell surface receptor (PRR) and the pathogencell surface ligand (PAMP) promotes relatively prompt phagocytosis ofthe pathogens by innate immune cells and also triggers the expression bythese host cells of the CD80 and CD86 surface proteins, which areimportant in recruiting the activation of the adaptive (acquired) immunesystem, over time, to generate an antigen-specific immune response.

Toll-like receptors (TLRs) are the PRRs of primary importance in highermammals: in the human innate immune system there are at least tendifferent TLR polypeptides, while in mice there are at least 11different TLRs (Rich, T., Toll and Toll-Like Receptors: An ImmunologicPerspective. 2010 Kluwer Academic/Plenum Publishers, Dordrecht, NL; seealso, e.g., Gorden et al., 2005 J. Immunol. 174:1259 and referencescited therein). These type I integral membrane proteins associate withadaptor proteins and, when engaged by appropriate ligands, mediatesignal transduction that results in activation of downstreamtranscription factors (e.g., NF-κB) to promote expression of cytokines,chemokines and other activation markers.

TLR2 is a TLR that binds primarily to potential pathogens such as Grampositive bacteria, Mycobacteria, Borrelia (spirochetes), and yeast. TLR2is present on the surfaces of certain host innate immune system cells,including myeloid lineage hematopoietic cells, as part of a heterodimerthat may occur (in association with TLR1) as a TLR1/2 heterodimer, or(in association with TLR6) as a TLR2/6 heterodimer. Some innate immunesystem cells, including myeloid lineage hematopoietic cells, alsoexpress TLR4, which binds Gram negative bacteria and also recognizes LPSand lipoteichoic acid. TLR4 occurs as a cell surface (TLR4/4) homodimer.The bacterial components recognized in binding interactions by TLR2 andTLR4 include peptidoglycan, lipoteichoic acid, and tripalmitoylatedlipoproteins. Interference with TLR2/6 heterodimer assembly impairedTLR2-mediated sepsis without affecting TLR4-mediated sepsis (Fink etal., 2013 J. Immunol. 190:6410); hence, TLR2-mediated sepsis andTLR4-mediated sepsis can proceed via mechanisms that are independent ofone another.

The pathophysiology of sepsis is complex but begins at a site ofinfection when a pathogen causes tissue injury, after first stimulatingthe innate immune system via TLRs and other PRRs, and subsequentlyprovoking a response by the adaptive (acquired) immune system. Besidesthe pathogen-derived components (PAMPs) that are locally released earlyin this sequence of inflammatory events, accompanying tissue damage mayalso lead to the release of host cellular components that activate theinnate immune response and are referred to as “danger associatedmolecular patterns”, or DAMPs. Examples of DAMPs include heat shockproteins (hsp) and alarmins, such as human mobility box group-1 protein.Coagulation factors, complement factors, mast cells, and platelets mayall also play a role in this initial phase of localized tissue injury,which can lead to a local inflammatory response.

The hallmarks of inflammation include one or more of heat, redness,swelling and pain to which vasodilators are significant contributors, asmediated, for example, by bradykinins and histamine. The inflammatoryresponse also involves the release of chemokines and activatedcomplement factors that influence migration of neutrophils into the areaof tissue injury and/or infection. Phagocytosis is enhanced by theinteraction between PAMPs and PRRs on the surfaces of innate immunecells, with the subsequent release by these cells of pro-inflammatorycytokines such as tumor necrosis factor-alpha (TNFα), interleukin-1(IL-1), interleukin-6 (IL-6), and interferon-gamma (IFNγ). The primarypurpose of the localized inflammatory process is to contain and destroythe pathogen. However if containment is not achieved and the pathogen isnot eradicated, then the inflammatory process and the infection mayspread. Once these events become systemic, the patient is at risk ofdeveloping sepsis (Hotchkiss et al., 2003 New Engl. J. Med. 348(2):138-150).

In systemic inflammation, the area of tissue injury that had previouslybeen contained to a local milieu now instead sheds necrotic cellularcomponents, pathogen and pathogen-derived debris, alarmins, PAMPs, andinflammatory cytokines into the circulation. The inflammatory processthus activates systemically many of the same innate immune system (e.g.,PAMP-, DAMP- and PRR-driven) processes that were once localized. Globalinflammatory phenomena characterize the resulting response, whichincludes systemic vasodilation, reduced vascular resistance, andincreased cardiac output with tachycardia and tachypnea. The edemacaused by vasodilation causes hypotension and hypoperfusion of criticalorgans. Ischemia in the organs promotes further tissue damage, which inturn further stimulates the inflammatory process. Concurrent with suchwidespread inflammatory events in sepsis, there is also evidence ofimmune suppression (manifest as lymphocyte anergy and theapoptosis-induced loss of CD4 T cells, B cells, and dendritic cells),along with reversible suspension of cellular functions in the organs(cell hibernation and cell stunning). These events contribute tosecondary and nosocomial infections, complicating the host's status andfurther stimulating the inflammatory process (Fry, 2012, The AmericanSurgeon 78:1-8).

Clinical trials of treating sepsis with various anti-inflammatoryproducts such as corticosteroids, anti-endotoxin antibodies, anti-TNFαproducts, and IL-1 receptor antagonists, have been conducted withoutsuccess (Hotchkiss et al., 2003 New Engl. J. Med. 348(2):138-150).

In a murine sepsis model, mice deficient for TLR2 or TLR2-derivedsignals were resistant to sepsis (Meng et al., 2004 J. Clin. Invest.113(10):1473-1481). Meng et al. (2004) described a monoclonal antibodyspecific for the murine TLR2 extracellular domain that was able to blockearly events in a sepsis model system by interfering with the binding ofa TLR2 agonist (P₃CSK4, a mimetic of tripalmitoylated proteins found onbacterial surfaces) to TLR2.

Although the monoclonal antibody of Meng cross-reacts with human TLR2(see also U.S. Pat. No. 8,623,353), this antibody is a murineimmunoglobulin and requires specific binding interactions contributed bythe immunoglobulin variable regions of two polypeptides (i.e., both theheavy and light chains). As such, problems associated with production ofa protein macromolecule having six defined murine complementaritydetermining regions (CDRs) that originate in two distinct polypeptides(CDRs 1-3 from each of the immunoglobulin heavy and light chains) mayhinder the development of a human therapeutic, such as proper foldingand assembly of the variable regions, and removal of immunogenicity byengineering out murine determinants without compromising antigen bindingspecificity. Additionally, the rodent model may be limited in itspredictive value for human sepsis, as noted by Decker (2004 J. Clin.Invest. 113:1387-1389).

A potential role for TLR2 in sepsis was also the subject of a report byNavarini et al. (2009 Proc. Nat. Acad. Sci. USA 106:7107-7112). Theseauthors described experiments in which the TLR2 ligand Pam2Cys(5-(2,3-bis(palmitoyloxy)propyl)cysteine), a Mycoplasma-derivedlipopeptide, elicited sepsis-like innate immune activation in a murinemodel of Listeria monocytogenes-driven neutrophil exhaustion.Co-administration to immunologically intact mice of an otherwisenon-lethal (low) dose of Listeria along with Pam2Cys resulted inoverwhelmingly lethal bacterial infections. Autopsy revealed depletionof neutrophils from bone marrow reservoirs (due to neutrophil migration)and the absence (due to neutrophil apoptosis) of live neutrophilinfiltrates from tissue sites which, in experimental control animalsthat did not receive Pam2Cys, were abundant in neutrophils. Micegenetically deficient for TLR2 (tlr2−/−), by contrast, were free ofdisease when subject to the same inoculation regimen, a result theauthors attributed to their lack of TLR2 receptors through which Pam2Cyscould initiate the inflammatory cascade.

Use of a TLR2 ligand, Pam2Cys, thus detrimentally escalated innateimmune activation that was instigated by a sub-lethal infection, causingit to progress to sepsis in much the same way as a lethal (high)Listeria dose: Neutrophils were driven to apoptosis without any apparentability to forestall the immunosuppressive cytokine profile elaboratedby phagocytes (macrophages and dendritic cells) involved in theirclearance, thereby favoring an overwhelming bacterial infection (Rogeret al., 2009 Proc. Nat. Acad. Sci. USA 106: 6889-6890). Multipleadditional reports describe significant neutrophil roles in thepathogenesis of sepsis, including exacerbation of disease by TLR2activation (Navarini et al., 2009 Proc. Nat. Acad. Sci. USA 106:7107;Roger et al., 2009 Proc. Nat. Acad. Sci. USA 106:6889; Alves-Filho etal., 2009 Proc. Nat. Acad. Sci. USA 106:4018; Zou et al., 2011 Shock36:370; Castoldi et al, 2012 PLoS ONE 7(5):e37584; Pene et al., 2009Infect. Immun. 77(12):5651).

Mice deficient for TLR4 or TLR4-derived signals were alsosepsis-resistant, including experimental animals treated with ananti-TLR antibody (Roger et al., 2009 Proc. Nat. Acad. Sci. USA106(7):2348) and animals that were genetically engineered to lack TLR2and TLR4 (Castoldi et al, 2012 PLoS ONE 7(5):e37584; Pene et al., 2009Infect. Immun. 77(12):5651). The TLR4 antagonist Eritoran, however,failed to demonstrate efficacy for treating sepsis in a recent clinicalstudy (Opal et al., 2013 J. Amer. Med. Assoc. 309(11):1154) despiteprevious encouraging reports for this agent (Tidswell et al., 2010 Crit.Care Med. 38:72; Barochia et al., 2011 Expert Opin. Drug Metab. Toxicol.7(4):479), and the anti-TLR4 antibody described by Roger et al. (2009)had no effect on TLR2-mediated sepsis.

Despite previous work pointing to exacerbation of sepsis that resultsfrom activating TLR2 and/or TLR4 (Navarini et al., 2009 Proc. Nat. Acad.Sci. USA 106:7107; Roger et al., 2009 Proc. Nat. Acad. Sci. USA106:6889; Alves-Filho et al., 2009 Proc. Nat. Acad. Sci. USA 106:4018;Zou et al., 2011 Shock 36:370; Castoldi et al, 2012 PLoS ONE7(5):e37584; Pene et al., 2009 Infect. Immun. 77(12):5651), efforts inthe art to date have failed to arrive at an effective TLR2- andTLR4-directed immunomodulator for sepsis (Lorne et al., 2010 IntensiveCare Med. 36:1826) that has a beneficial mechanism of action andfavorable efficacy profile, and that further offers ease of manufactureand is nonimmunogenic in humans.

Clearly there remains a need for improved compositions and methods totreat, reduce the severity of, or reduce the likelihood of occurrence ofTLR2- and/or TLR4-mediated sepsis. Certain of the presently disclosedinvention embodiments address this need and provide other relatedadvantages.

Cancer

Cancer remains a devastating and largely intractable disease withsignificant unmet needs in the areas of patient treatment, clinicaloutcome and overall survival. The American Cancer Society (ACS)estimates that there will be over 1.6 million new cases of cancerdiagnosed in the United States in 2012, not including non-invasivecarcinoma in situ and also not including new cases of basal cellcarcinoma and squamous cell skin cancer. The ACS also projects thatthere will be 577,190 cancer-related deaths in 2012, or an average of1500 Americans dying each day from cancer, making cancer the second mostcommon cause of death in the U.S., after heart disease. In 2007 thetotal medical cost of cancer in the U.S. was $226.8 billion, includingdirect medical costs for treatment of $103.8 billion and indirect costsdue to lost productivity and premature death of $123 billion. The meanfive-year survival rate overall for U.S. cancer patients has improvedfrom 49% in 1975-1977 to 67% in 2001-2007. There clearly, however, stillremains a pressing need for improved treatments and enhanced overalloutcomes for cancer patients.

Transplantation

Organ transplantation is often the best or only treatment option forend-stage organ failure, such as kidney disease, chronic conditions suchas severe cirrhosis of the liver, and cancer, such as liver cancer,leukemias and lymphomas. Both solid organ and bone marrow transplantsare performed in order to treat patients in need. It is estimated thatabout 100,000 solid organ transplants were performed worldwide in 2007,and of the roughly 30,000 bone marrow transplants performed annually,about 15,000 are allogenic transplants. Kidney, liver and hearttransplants are the most common solid organ transplants.

The worldwide demand for donor tissues and organs far surpasses thesupply, and there is a significant need in transplantation medicine toimprove the availability of donor organs and minimize the long term riskof rejection. For example, the World Health Organization (WHO) hasestimated that only 10% of those in need of kidney transplants manage toget one. The National Kidney Foundation claims that about 18 patientsdie daily while waiting for a transplant of a vital organ such as aheart, liver, kidney, pancreas, lung, or bone marrow. Furthermore thelong-term graft survival rates for kidney and liver transplants areabout 60-70% at 5-10 years post-transplant.

Rejection of the donor tissue by the host immune system is one of thelargest problems faced in allogenic transplants. Despite donor-recipienthuman leukocyte antigen (HLA) histocompatibility matching and ABO bloodgroup testing to provide matched donor tissue to the recipient, patientsreceiving transplants must undergo immunosuppressive treatment in aneffort to prevent graft rejection or, in the case of bone marrowtransplants, graft-versus-host disease (GVHD). Most immunosuppressorstarget T cells or cytokines secreted by T cells, and types ofimmunosuppressive agents currently used include monoclonal antibodies tolymphocytes and cytokine receptors (e.g., anti-IL-2Rα), calcineurininhibitors (e.g., cyclosporine and tacrolimus), and cytokine receptorsignal transduction inhibitors (e.g., sirolimus) (Chinen and Buckley, JAllergy Clin Immunol, 2010, 125(2 Suppl 2):S324-S335). The downside tousing immunosuppressive agents, sometimes over a course of severalmonths, is that while protecting the graft from being rejected by thehost immune system, or vice versa in the case of GVHD, they make therecipient especially vulnerable to infections and malignancies.

In addition, despite advances in immunosuppressive treatments which havesignificantly improved first year graft survival, long-term survival isstill unsatisfactory. In a study of long term kidney graft survival,Fernandex-Rodriguez et al (Transplantation Proceedings 2009 41(6):2357-2359) followed 1,029 first renal transplantations performed betweenNovember 1979 and December 2007, observed renal graft survival at 1, 5and 10 years and correlated the results to the immunosuppressive therapyused, including azathioprine (AZA), cyclosporine (CsA), and tacrolimus(TAC). The findings indicated that graft survival rates at 5 and 10years post-transplant were, respectively, 56% and 46% on AZA, 69% and54% on CsA, and 77% and 60% on TAC. The study concluded that despite thedecrease in acute rejection in kidney transplants, there was asignificant decrease in renal graft survival after 12 months. Anotherstudy by Ruiz et al (Arch Surg 2006 141:735-742) reported liver graftsurvival at 1, 3 and 5 years post-transplant of 70%, 65% and 65%,respectively, and kidney graft survival at 1, 3 and 5 yearspost-transplant of 76%, 72% and 70%, respectively, thereby demonstratingthat there is a significant decrease in graft survival following thefirst year.

As a semi-allograft, the maternal-host acceptance and tolerance of anembryo and placenta is similar in many ways to an allogeneic organtransplant. Implantation of the blastocyst into the uterine wall is acritical checkpoint for a successful pregnancy and results in the embryoadhering to the uterine lining and generating a vascular connection.Implantation failure can result in repeated miscarriages and failed invitro fertilization (IVF) attempts. In particular, embryo implantationsuccess rates in IVF patients vary widely, and success rates rangingfrom about 15% to about 30% have been reported (see, e.g., Croo et al.Human Reproduction, 15(6):1383-1388, 2000). Inflammation and themother's immune response are believed to play a role in the successfulimplantation of an embryo.

Inflammation also plays a role later on in pregnancy, such as inpreeclampsia and eclampsia, which affect an estimated 5-8% of allpregnancies and are the leading cause of maternal and fetal illness andmortality worldwide. Preeclampsia is characterized by high bloodpressure and proteinuria, and if left unchecked, it can lead to theseizures of eclampsia. During pregnancy, the maternal adaptive immuneresponse is down-regulated, and the innate immune response is enhanced.However, the innate immunity also must be regulated, and it has beenshown that neutrophils, non-antigen specific white blood cells ofhematopoietic origin that are typically associated with inflammatory andanti-microbial responses, play a large role in preeclampsia (Cadden andWalsh, Hypertens Pregnancy (2008) 27(4):396-405).

Indeed, studies indicate that innate immune cells, such as neutrophils(often referred to as polymorphonuclear neutrophils, or PMN), areimportant in shaping, enhancing and regulating the adaptive immuneresponse (see, e.g., Soehnlein, Trends in Immunology, 2009,30(11):511-512 and Müller et al, Trends in Immunology, 2009,30(11):522-530). Recent studies indicate that neutrophils may play asignificant role in antitumor reactions (DiCarlo et al., 2001 Blood97:339; Mumm et al., 2011 Canc. Cell 20:781) and in transplantrejection, in addition to the historical role of lymphocytes. Inparticular, Soo et al. (J. Heart and Lung Transplantation, 2009,28(11):1198-1205) examined pre-operative neutrophil adhesion moleculeexpression after in vitro stimulation with LPS or PMA and thencorrelated these results with actual allograft success. Interestingly,pre-operative neutrophil surface CD11 b expression after LPS stimulationcorrelated proportionally with the degree of rejection as detected inthe first endomyocardial biopsy sample post heart transplantation, andthe authors concluded “that neutrophils may contribute more to cardiacallograft rejection than previously thought.”

Another study identified increased IL-17 and neutrophilia in thebroncho-alveolar lavage of patients undergoing acute lung transplantrejection (Chest 2007 131(6):1988-9). Furthermore, neutrophils have beenshown to play a significant role in xenotransplantation rejection(Transplant Immunology 2009 21:70-74, Transplantation 2004 78:1721-1718), and means of attenuating neutrophil activity may play asignificant role in improving the success of this type oftransplantation approach. There is also increasing evidence thatneutrophils can be activated to express MHC Class II molecules anddevelop antigen presenting cell (APC) characteristics and releasecytokines such as IL-4, IL6, IL-10, IL-12, and TNFa and suppress T cellactivity (e.g., Muller et al., 2009 Trends in Immunology 30(11):522-530,2009; Vasconcelos et al., 2006 Blood 107:2192-2199; Rodriguez et al.,2009 Cancer Research 69:1553-1560, 2009. Therefore the role ofneutrophils may also be involved in allograft and xenograft acceptance.

Neutrophils are clearly of interest for research due to their role ininflammation and infection as well as other immune functions, includingroles in pregnancy, transplantation and autoimmunity. A number of cellsurface markers present on neutrophils have been utilized in an effortto characterize, identify and determine their activation state, such asCD64, CD11b, and CD83; however few neutrophil markers exist that canreadily be used to identify a distinct subset of neutrophils in the sameway that other hematopoietic cell surface markers can be used tocategorize adaptive immune cells, for instance, according tomaturational state, differentiation lineage, and functional properties(e.g., CD4 versus CD8 T lymphocytes, surface immunoglobulin (sIg)changes in B lymphocyte differentiation and maturation, and otherregulatory cell markers, e.g., CD45 isoforms, distinct integrin α and βchain heterodimer expression, etc.). Accordingly, a need exists for morerefined neutrophil markers in order to functionally characterize thesecells. (See, e.g., Mason et al., (Eds.), Leukocyte Typing VII, 2002Oxford Univ. Press, USA.)

An increased understanding of the mechanisms resulting in graftrejection have lead to advancements in the availability and mechanisticunderstanding of immunosuppressants; however, there is still asignificant medical need for immunosuppressive agents that are capableof improving the acceptance of donor organs and minimizing the risk ofrejection while at the same time placing recipients at a lesser risk ofinfections and malignancies. In particular, there is an unmet need fortreatments that improve long term graft survival and also improve thesuccess of xenotransplantation. In addition, immunosuppressive agentsthat are useful for conception, providing improved rates of implantationand a reduced risk of multiples, as well as preventing conditions likepreeclampsia, are needed.

The compositions and methods of the present invention address the needsdescribed above and offer other related advantages.

BRIEF SUMMARY

According to certain embodiments of the invention disclosed herein,there is provided an isolated immunomodulatory polypeptide of no morethan 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13 or 12 amino acids, which comprises either (i) the amino acidsequence KSIAYLQMNSLK as set forth in SEQ ID NO:2, or (ii) the aminoacid sequence of general formula:

K-X1-X2-X3-YLQM-X4-X5-LK as set forth in SEQ ID NO:106, wherein X1 isselected from S and N, X2 is selected from I, T, S, M, R and N, X3 isselected from A, L, V and Q, X4 is selected from N, D, S, T and A, andX5 is selected from S, T and N.

In certain further embodiments at least one of: (a) the immunomodulatorypolypeptide comprises up to 23 contiguous amino acids of the amino acidsequence set forth in any one of SEQ ID NOS:3 and 5-104, said 23contiguous amino acids including the amino acid sequence KSIAYLQMNSLK asset forth in SEQ ID NO:2, or (b) the immunomodulatory polypeptidecomprises no more than 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12contiguous amino acids. In certain embodiments there is provided apharmaceutical composition, comprising any one or more of the abovedescribed immunomodulatory polypeptides; and (b) a physiologicallyacceptable carrier. In certain other embodiments there is provided afusion protein comprising any one of the above describedimmunomodulatory polypeptides fused to a fusion polypeptide domain. Incertain embodiments there is provided a pharmaceutical compositioncomprising this fusion protein; and a physiologically acceptablecarrier.

According to certain other embodiments there is provided animmunomodulatory polypeptide of no more than 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 amino acids for useas a medicament, wherein the immunomodulatory polypeptide compriseseither (i) the amino acid sequence KSIAYLQMNSLK as set forth in SEQ IDNO:2, or (ii) the amino acid sequence of general formula:K-X1-X2-X3-YLQM-X4-X5-LK as set forth in SEQ ID NO:106, wherein X1 isselected from S and N, X2 is selected from I, T, S, M, R and N, X3 isselected from A, L, V and Q, X4 is selected from N, D, S, T and A, andX5 is selected from S, T and N.

In another embodiment there is provided an immunomodulatory polypeptideof no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13 or 12 amino acids for use in the treatment ofTLR2-mediated sepsis and/or TLR4-mediated sepsis, wherein theimmunomodulatory polypeptide comprises either (i) the amino acidsequence KSIAYLQMNSLK as set forth in SEQ ID NO:2, or (ii) the aminoacid sequence of general formula: K-X1-X2-X3-YLQM-X4-X5-LK as set forthin SEQ ID NO:106, wherein X1 is selected from S and N, X2 is selectedfrom I, T, S, M, R and N, X3 is selected from A, L, V and Q, X4 isselected from N, D, S, T and A, and X5 is selected from S, T and N.

In another embodiment there is provided an immunomodulatory polypeptideof no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13 or 12 amino acids for use in the treatment of graftrejection to be decreased in a graft transplant recipient, wherein theimmunomodulatory polypeptide comprises either (i) the amino acidsequence KSIAYLQMNSLK as set forth in SEQ ID NO:2, or (ii) the aminoacid sequence of general formula: K-X1-X2-X3-YLQM-X4-X5-LK as set forthin SEQ ID NO:106, wherein X1 is selected from S and N, X2 is selectedfrom I, T, S, M, R and N, X3 is selected from A, L, V and Q, X4 isselected from N, D, S, T and A, and X5 is selected from S, T and N.

In another embodiment there is provided an immunomodulatory polypeptideof no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13 or 12 amino acids for use in the treatment of graftrejection to be decreased in a graft transplant recipient graftrejection to be decreased in a graft transplant recipient, wherein thegraft transplant is selected from kidney, heart, liver, pancreas andlung, wherein the immunomodulatory polypeptide comprises either (i) theamino acid sequence KSIAYLQMNSLK as set forth in SEQ ID NO:2, or (ii)the amino acid sequence of general formula: K-X1-X2-X3-YLQM-X4-X5-LK asset forth in SEQ ID NO:106, wherein X1 is selected from S and N, X2 isselected from I, T, S, M, R and N, X3 is selected from A, L, V and Q, X4is selected from N, D, S, T and A, and X5 is selected from S, T and N.

In another embodiment there is provided an immunomodulatory polypeptideof no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13 or 12 amino acids for use in the treatment of graftversus host disease in a bone marrow transplant recipient, wherein theimmunomodulatory polypeptide comprises either (i) the amino acidsequence KSIAYLQMNSLK as set forth in SEQ ID NO:2, or (ii) the aminoacid sequence of general formula: K-X1-X2-X3-YLQM-X4-X5-LK as set forthin SEQ ID NO:106, wherein X1 is selected from S and N, X2 is selectedfrom I, T, S, M, R and N, X3 is selected from A, L, V and Q, X4 isselected from N, D, S, T and A, and X5 is selected from S, T and N.

In another embodiment there is provided an immunomodulatory polypeptideof no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13 or 12 amino acids for use in the treatment ofpreeclampsia, severe preeclampsia or hemolysis-elevated liverenzymes-low platelet count (HELLP) syndrome, wherein theimmunomodulatory polypeptide comprises either (i) the amino acidsequence KSIAYLQMNSLK as set forth in SEQ ID NO:2, or (ii) the aminoacid sequence of general formula: K-X1-X2-X3-YLQM-X4-X5-LK as set forthin SEQ ID NO:106, wherein X1 is selected from S and N, X2 is selectedfrom I, T, S, M, R and N, X3 is selected from A, L, V and Q, X4 isselected from N, D, S, T and A, and X5 is selected from S, T and N.

In another embodiment there is provided an immunomodulatory polypeptideof no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13 or 12 amino acids for use in the treatment ofrheumatoid arthritis, wherein the immunomodulatory polypeptide compriseseither (i) the amino acid sequence KSIAYLQMNSLK as set forth in SEQ IDNO:2, or (ii) the amino acid sequence of general formula:K-X1-X2-X3-YLQM-X4-X5-LK as set forth in SEQ ID NO:106, wherein X1 isselected from S and N, X2 is selected from I, T, S, M, R and N, X3 isselected from A, L, V and Q, X4 is selected from N, D, S, T and A, andX5 is selected from S, T and N.

In another embodiment there is provided an immunomodulatory polypeptideof no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13 or 12 amino acids for use in the treatment of amalignant condition, wherein the immunomodulatory polypeptide compriseseither (i) the amino acid sequence KSIAYLQMNSLK as set forth in SEQ IDNO:2, or (ii) the amino acid sequence of general formula:K-X1-X2-X3-YLQM-X4-X5-LK as set forth in SEQ ID NO:106, wherein X1 isselected from S and N, X2 is selected from I, T, S, M, R and N, X3 isselected from A, L, V and Q, X4 is selected from N, D, S, T and A, andX5 is selected from S, T and N.

In another embodiment there is provided an immunomodulatory polypeptideof no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13 or 12 amino acids for use in the treatment of amalignant condition, wherein the immunomodulatory polypeptide compriseseither (i) the amino acid sequence KSIAYLQMNSLK as set forth in SEQ IDNO:2, or (ii) the amino acid sequence of general formula:K-X1-X2-X3-YLQM-X4-X5-LK as set forth in SEQ ID NO:106, wherein X1 isselected from S and N, X2 is selected from I, T, S, M, R and N, X3 isselected from A, L, V and Q, X4 is selected from N, D, S, T and A, andX5 is selected from S, T and N. In certain further embodiments, themalignant condition is selected from breast cancer, ovarian cancer,adenoma, colorectal carcinoma, gastric carcinoma, lung carcinoma,prostate carcinoma, hepatocellular carcinoma, melanoma, leukemia andlymphoma.

In another embodiment there is provided an immunomodulatory polypeptideof no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13 or 12 amino acids for use in the treatment of anautoimmune disease, wherein the immunomodulatory polypeptide compriseseither (i) the amino acid sequence KSIAYLQMNSLK as set forth in SEQ IDNO:2, or (ii) the amino acid sequence of general formula:K-X1-X2-X3-YLQM-X4-X5-LK as set forth in SEQ ID NO:106, wherein X1 isselected from S and N, X2 is selected from I, T, S, M, R and N, X3 isselected from A, L, V and Q, X4 is selected from N, D, S, T and A, andX5 is selected from S, T and N. In certain further embodiments, theautoimmune disease is selected from rheumatoid arthritis, psoriaticarthritis, ulcerative colitis, Crohn's disease, seronegativespondyloarthopathies, systemic lupus erythematosus, Behcet's disease andvasculitis.

Turning to another embodiment of the invention described herein, thereis provided a method of inducing a peripheral blood white cell responsethat includes cellular release of at least one of IL-6, IL-10 and TNFα,comprising contacting one or a plurality of peripheral blood white cellsin vitro or in vivo with the above-described immunomodulatorypolypeptide, under conditions and for a time sufficient to inducedetectable cellular release of at least one of IL-6, IL-10 and TNFα. Inanother embodiment there is provided a method of treating an organ to betransplanted into an allogeneic recipient to reduce a likelihood orseverity of allograft rejection by the recipient, comprising contactingthe organ with the above-described immunomodulatory polypeptide, underconditions and for a time sufficient to reduce the likelihood orseverity of allograft rejection.

In certain other embodiments there is provided a method of selectivelylabeling a mammalian peripheral blood white cell neutrophilsubpopulation, comprising contacting a population of mammalianperipheral blood white cells which comprises neutrophils with the abovedescribed immunomodulatory polypeptide, wherein either (i) theimmunomodulatory polypeptide comprises a detectable label or (ii) theimmunomodulatory polypeptide is indirectly detected. In certain furtherembodiments the detectable label is selected from the group consistingof a fluorescent dye, a radioactive substance and a metal particle.

In certain other embodiments there is presently provided a method fortreating, reducing severity of, or reducing likelihood of occurrence ofTLR2-mediated sepsis and/or TLR4-mediated sepsis in a subject,comprising administering to the subject a therapeutically effectiveamount of an immunomodulatory polypeptide that comprises either theamino acid sequence set forth in SEQ ID NO:2 or the amino acid sequenceset forth in SEQ ID NO:106.

In certain other embodiments there is provided a method of treating apatient, comprising administering to the patient a therapeuticallyeffective amount of an immunomodulatory polypeptide that compriseseither the amino acid sequence set forth in SEQ ID NO:2 or the aminoacid sequence set forth in SEQ ID NO:106, wherein the method is selectedfrom: (a) a method for treating, reducing severity of, or reducinglikelihood of occurrence of TLR2-mediated sepsis in the patient, (b) amethod of decreasing graft rejection wherein the patient is a transplantpatient, (c) the method of (b) wherein the transplant is selected fromkidney, heart, liver, pancreas and lung, (d) a method of treating ordecreasing graft versus host disease wherein the patient is a bonemarrow transplant patient, (e) a method of treating preeclampsia orhemolysis-elevated liver enzymes-low platelet count (HELLP) syndrome inthe patient, (f) the method of (e) wherein the patient has beendiagnosed with severe preeclampsia, (g) a method of treating rheumatoidarthritis in the patient, (h) a method of treating a malignant conditionin the patient, (i) the method of (h) wherein treating the malignantcondition comprises at least one of killing a tumor cell and inhibitingmetastasis, (j) the method of (h) wherein the malignant condition isselected from breast cancer, ovarian cancer, adenoma, colorectalcarcinoma, gastric carcinoma, lung carcinoma, prostate carcinoma,hepatocellular carcinoma, melanoma, leukemia and lymphoma, (k) a methodof treating an autoimmune disease in the patient, and (l) the method of(k) wherein the autoimmune disease is selected from rheumatoidarthritis, psoriatic arthritis, ulcerative colitis, Crohn's disease,seronegative spondyloarthopathies, systemic lupus erythematosus,Behcet's disease and vasculitis.

According to certain further embodiments of the above describedimmunomodulatory polypeptide the above described methods, theimmunomodulatory polypeptide is selected from: (a) an immunomodulatorypolypeptide of no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13 or 12 amino acids which comprises theamino acid sequence KSIAYLQMNSLK as set forth in SEQ ID NO:2, (b) animmunomodulatory peptide that is selected from the group consisting ofIgX, a fragment of IgX, and a variant of IgX, (c) an immunomodulatorypolypeptide that comprises a scFv of an immunoglobulin that is selectedfrom (i) IgX and (ii) an immunoglobulin that comprises an immunoglobulinpolypeptide that is selected from the group consisting of a polypeptidecomprising the amino acid sequence set forth in any one of SEQ ID NOS:3and 5-104, (d) an immunomodulatory polypeptide that comprises a Fab ofan immunoglobulin that is selected from (i) IgX and (ii) animmunoglobulin that comprises an immunoglobulin polypeptide that isselected from the group consisting of a polypeptide comprising the aminoacid sequence set forth in any one of SEQ ID NOS:3 and 5-104, (e) animmunomodulatory polypeptide that comprises a (Fab′)₂ of animmunoglobulin that is selected from (i) IgX and (ii) an immunoglobulinthat comprises an immunoglobulin polypeptide that is selected from thegroup consisting of a polypeptide comprising the amino acid sequence setforth in any one of SEQ ID NOS:3 and 5-104, (f) an immunomodulatorypolypeptide that comprises an intact immunoglobulin heavy chain variableregion having an amino acid sequence of an immunoglobulin variableregion that is present in an immunoglobulin polypeptide that is selectedfrom the group consisting of a polypeptide comprising the amino acidsequence set forth in any one of SEQ ID NOS:3 and 5-104, (g) animmunomodulatory polypeptide that comprises an intact immunoglobulinheavy chain having an amino acid sequence of an immunoglobulin heavychain that is selected from the group consisting of the sequences setforth in SEQ ID NOs:3 and 5-104, and (h) an immunomodulatory polypeptidethat comprises an intact antibody, wherein the antibody comprises anintact immunoglobulin heavy chain having an amino acid sequence of animmunoglobulin heavy chain that is selected from the group consisting ofthe sequences set forth in SEQ ID NOs: 3 and 5-104.

Turning to another embodiment, there is provided an isolatedpolynucleotide comprising a nucleic acid sequence that encodes the abovedescribed immunomodulatory polypeptide, and in another embodiment thereis provided an expression vector comprising said polynucleotide, and inanother embodiment there is provided a host cell transformed ortransfected with said expression vector. In certain embodiments there isprovided a method of producing an immunomodulatory polypeptide of nomore than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13 or 12 amino acids that comprises either (i) the aminoacid sequence set forth in SEQ ID NO:2 or (ii) the amino acid sequenceset forth in SEQ ID NO:106, said method comprising the steps of a)culturing the host cell described above under conditions and for a timesufficient to permit expression of the immunomodulatory polypeptide; andb) isolating the immunomodulatory polypeptide from the cultured hostcell.

In another embodiment there is provided an isolated polynucleotide thatis selected from: (a) an isolated antisense polynucleotide comprising anucleic acid sequence that is complementary to the polynucleotidedescribed above which encodes the immunomodulatory polypeptide, (b) anisolated small interfering RNA (siRNA) polynucleotide that is capable ofsubstantially silencing, and is complementary to a region of at least 18and no more than 69 contiguous nucleotides in, a nucleic acid whichencodes the immunomodulatory polypeptide described above, and (c) anisolated ribozyme that specifically binds to the polynucleotidedescribed above which encodes the immunomodulatory polypeptide.

According to another embodiment there is provided an isolated antibody,or antigen-binding fragment thereof, that specifically binds to theimmunomodulatory polypeptide described above, which in certainembodiments is a monoclonal antibody. Certain embodiments provide apharmaceutical composition comprising the just-described antibody and aphysiologically acceptable carrier.

In another embodiment there is provided a method for detecting, in abiological sample, an immunomodulatory polypeptide that comprises eitherthe amino acid sequence set forth in SEQ ID NO:2 or the amino acidsequence set forth in SEQ ID NO:106, said method comprising the stepsof: (a) contacting the biological sample with an antibody thatspecifically binds said immunomodulatory polypeptide, or anantigen-binding fragment of said antibody, under conditions and for atime sufficient for specific antibody binding to the immunomodulatorypolypeptide to take place; and (b) detecting specific binding of theantibody to the immunomodulatory peptide, and thereby detecting theimmunomodulatory peptide in the sample. In certain further embodimentsat least one of: (i) the antibody is linked to a support material, (ii)the antibody is linked to a detectable label, or (iii) the biologicalsample is obtained from a subject that is selected from a human, anon-human primate, a non-primate mammal, a non-mammalian vertebrate, aninvertebrate eukaryote and a prokaryote.

In certain other embodiments there is provided a method of promotingimplantation of an embryo in a pregnant or pseudopregnant mammal,comprising contacting at least one of the embryo and the pregnant orpseudopregnant mammal with an immunomodulatory polypeptide thatcomprises either the amino acid sequence set forth in SEQ ID NO:2 or theamino acid sequence set forth in SEQ ID NO:106, under conditions and fora time sufficient to promote embryonic implantation. In certain furtherembodiments the pregnant or pseudopregnant mammal is a human, and incertain embodiments the embryo is produced by in vitro fertilization.

In another embodiment there is provided a method for detecting, in abiological sample that comprises one or a plurality of nucleic acidmolecules, expression of a polynucleotide that encodes animmunomodulatory polypeptide that comprises either the amino acidsequence set forth in SEQ ID NO:2 or the amino acid sequence set forthin SEQ ID NO:106, said method comprising the steps of: (a) contactingthe sample with at least one of (i) the antisense polynucleotidedescribed above, and (ii) the polynucleotide described above whichcomprises a nucleic acid sequence that encodes the above describedimmunomodulatory polypeptide, under conditions and for a time sufficientfor specific nucleic acid hybridization to occur; and (b) detectingspecific hybridization of at least one nucleic acid of the sample to atleast one of said antisense polynucleotide and said immunomodulatorypolypeptide-encoding polynucleotide, and thereby detecting, in thesample, expression of the polynucleotide that encodes theimmunomodulatory peptide. In certain embodiments the biological sampleis obtained from a subject that is selected from a human, a non-humanprimate, a non-primate mammal, a non-mammalian vertebrate, aninvertebrate eukaryote and a prokaryote.

According to certain embodiments there is provided an isolatedimmunomodulatory polypeptide that competes with PeptideX2 or a variantthereof for specific binding to a human neutrophil, wherein saidPeptideX2 comprises the amino acid sequence set forth in SEQ ID NO:2 andwherein said variant thereof comprises the amino acid sequence set forthin SEQ ID NO:106. In certain further embodiments the immunomodulatorypolypeptide that competes with PeptideX2 or a variant thereof forspecific binding to a human neutrophil, interferes with PeptideX2binding to either or both of human TLR2 and human TLR4.

According to certain embodiments of the invention disclosed herein,there is provided an isolated immunomodulatory polypeptide of no morethan 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13 or 12 amino acids, which comprises either (i) the amino acidsequence KSIAYLQMNSLK as set forth in SEQ ID NO:2, or (ii) the aminoacid sequence of general formula: K-X1-X2-X3-YLQM-X4-X5-LK as set forthin SEQ ID NO:106, wherein X1 is selected from S and N, X2 is selectedfrom I, T, S, M, R and N, X3 is selected from A, L, V and Q, X4 isselected from N, D, S, T and A, and X5 is selected from S, T and N. Incertain further embodiments the polypeptide comprises up to 23contiguous amino acids of the amino acid sequence set forth in any oneof SEQ ID NOS:3 and 5-104, said 23 contiguous amino acids including theamino acid sequence KSIAYLQMNSLK as set forth in SEQ ID NO:2. In certainother further embodiments the immunomodulatory polypeptide of claim 1which comprises no more than 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or12 contiguous amino acids. In another embodiment there is provided anisolated polynucleotide comprising a nucleic acid sequence that encodesany of the just-described immunomodulatory polypeptides. In certainother embodiments there is provided an expression vector comprising thepolynucleotide. In certain other embodiments there is provided a hostcell transformed or transfected with the expression vector.

In certain other embodiments there is provided a method of producing animmunomodulatory polypeptide of no more than 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 amino acids thatcomprises the amino acid sequence set forth in SEQ ID NO:2 or in SEQ IDNO: 106, the method comprising the steps of a) culturing the abovedescribed host cell under conditions and for a time sufficient to permitexpression of the immunomodulatory polypeptide; and b) isolating theimmunomodulatory polypeptide from the cultured host cell. In anotherembodiment there is provided a pharmaceutical composition, comprising a)an immunomodulatory polypeptide of no more than 31, 30, 29, 28, 27, 26,25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 amino acids,comprising the amino acid sequence set forth in either SEQ ID NO:2 or inSEQ ID NO:106; and b) a physiologically acceptable carrier. In anotherembodiment there is provided an isolated antisense polynucleotidecomprising a nucleic acid sequence that is complementary to thepolynucleotide described above and herein. In another embodiment thereis provided an isolated small interfering RNA (siRNA) polynucleotidethat is capable of substantially silencing, and is complementary to aregion of 18-69 contiguous nucleotides in, a nucleic acid which encodesthe immunomodulatory polypeptide that comprises up to 23 contiguousamino acids of the amino acid sequence set forth in any one of SEQ IDNOS:3 and 5-104, said 23 contiguous amino acids including the amino acidsequence KSIAYLQMNSLK as set forth in SEQ ID NO:2. Certain otherembodiments provide an isolated ribozyme that specifically binds to thepolynucleotide described above and herein.

Certain other embodiments provide an isolated antibody, orantigen-binding fragment thereof, that specifically binds to animmunomodulatory polypeptide of no more than 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 amino acids, theimmunomodulatory polypeptide comprising the amino acid sequence setforth in SEQ ID NO:2 or in SEQ ID NO:106. In certain embodiments theantibody is a monoclonal antibody.

In certain embodiments there is provided a pharmaceutical compositioncomprising the just-described antibody and a physiologically acceptablecarrier. According to certain embodiments there is provided a method fordetecting, in a biological sample, an immunomodulatory polypeptide of nomore than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13 or 12 amino acids that comprises the amino acid sequenceset forth in SEQ ID NO:2 or in SEQ ID NO:106, said method comprising thesteps of: a) contacting the biological sample with an antibody thatspecifically binds said immunomodulatory polypeptide, or anantigen-binding fragment of said antibody, under conditions and for atime sufficient for specific antibody binding to the immunomodulatorypolypeptide to take place; and b) detecting specific binding of theantibody to the immunomodulatory peptide, and thereby detecting theimmunomodulatory peptide in the sample. In certain further embodimentsthe antibody is linked to a support material. In certain other furtherembodiments the antibody is linked to a detectable label. In certainother further embodiments the biological sample is obtained from asubject that is selected from a human, a non-human primate, anon-primate mammal, a non-mammalian vertebrate, an invertebrateeukaryote and a prokaryote.

In certain embodiments there is provided a pharmaceutical composition,comprising a) an immunomodulatory polypeptide of no more than 31, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12amino acids comprising the amino acid sequence set forth in SEQ ID NO:2or in SEQ ID NO:106; and b) a physiologically acceptable carrier.

In certain embodiments there is provided a method for detecting, in abiological sample that comprises one or a plurality of nucleic acidmolecules, expression of a polynucleotide that encodes animmunomodulatory polypeptide of no more than 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 amino acids,comprising the amino acid sequence set forth in SEQ ID NO:2 or in SEQ IDNO:106, said method comprising the steps of a) contacting the samplewith at least one of (i) the antisense polynucleotide of claim 9, and(ii) the polynucleotide of claim 4, under conditions and for a timesufficient for specific nucleic acid hybridization to occur; and b)detecting specific hybridization of at least one nucleic acid to atleast one of said antisense polynucleotide and said polynucleotide ofclaim 4, and thereby detecting, in the sample, expression of thepolynucleotide that encodes the immunomodulatory peptide. In certainfurther embodiments the biological sample is obtained from a subjectthat is selected from a human, a non-human primate, a non-primatemammal, a non-mammalian vertebrate, an invertebrate eukaryote and aprokaryote.

In another embodiment there is provided a fusion protein comprising animmunomodulatory polypeptide that comprises up to 23 contiguous aminoacids of the amino acid sequence set forth in any one of SEQ ID NOS:3and 5-104, said 23 contiguous amino acids including the amino acidsequence KSIAYLQMNSLK as set forth in SEQ ID NO:2 or the amino acidsequence set forth in SEQ ID NO:106, fused to a fusion polypeptidedomain. In certain further embodiments there is provided apharmaceutical composition comprising the fusion protein; and aphysiologically acceptable carrier.

In another embodiment there is provided a method of treating orpreventing graft rejection in a transplant patient, comprisingadministering a therapeutically effective amount of an immunomodulatorypolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2or in SEQ ID NO:106. In certain embodiments the transplant is selectedfrom kidney, heart, liver, pancreas and lung. In another embodimentthere is provided a method of treating or preventing graft versus hostdisease in a bone marrow transplant patient, comprising administering atherapeutically effective amount of an immunomodulatory polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:2 or in SEQ IDNO:106. In another embodiment there is provided a method of treatingpreeclampsia in a patient, comprising administering a therapeuticallyeffective amount of an immunomodulatory polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:2 or in SEQ ID NO:106. In anotherembodiment there is provided a method of treating or preventingeclampsia in a patient, comprising administering a therapeuticallyeffective amount of an immunomodulatory polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:2 or in SEQ ID NO:106. In certainfurther embodiments the patient has been diagnosed with severepreeclampsia. In another embodiment there is provided a method oftreating or preventing HELLP syndrome in a patient, comprisingadministering a therapeutically effective amount of an immunomodulatorypolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2or in SEQ ID NO:106. In certain further embodiments, the patient hasbeen diagnosed with severe preeclampsia.

In another embodiment there is provided a method of treating rheumatoidarthritis in a patient, comprising administering a therapeuticallyeffective amount of an immunomodulatory polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:2 or in SEQ ID NO:106.

In another embodiment there is provided a method of inducing aperipheral blood white cell response that includes cellular release ofat least one of IL-6, IL-10 and TNFα, comprising contacting one or aplurality of peripheral blood white cells in vitro or in vivo with animmunomodulatory polypeptide comprising the amino acid sequence setforth in SEQ ID NO:2 or in SEQ ID NO:106, under conditions and for atime sufficient to induce detectable cellular release of at least one ofIL-6, IL-10 and TNFα. In another embodiment there is provided a methodof treating an organ to be transplanted into an allogeneic recipient toreduce a likelihood or severity of allograft rejection by the recipient,comprising contacting the organ with an immunomodulatory polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:2 or in SEQ IDNO:106, under conditions and for a time sufficient to reduce thelikelihood or severity of allograft rejection. In another embodimentthere is provided a method of promoting implantation of an embryo in apregnant or pseudopregnant mammal, comprising contacting at least one ofthe embryo and the pregnant or pseudopregnant mammal with animmunomodulatory polypeptide comprising the amino acid sequence setforth in SEQ ID NO:2 or in SEQ ID NO:106, under conditions and for atime sufficient to promote embryonic implantation. In certain furtherembodiments the pregnant or pseudopregnant mammal is a human. In certainfurther embodiments the embryo is produced by in vitro fertilization.

In certain embodiments there is provided a method of selectivelylabeling a mammalian peripheral blood white cell neutrophilsubpopulation, comprising contacting a population of mammalianperipheral blood white cells which comprises neutrophils with animmunomodulatory polypeptide that comprises an immunomodulatorypolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2or in SEQ ID NO:106, wherein the immunomodulatory polypeptide comprisesa detectable label. In certain further embodiments the detectable labelis selected from the group consisting of a fluorescent dye, aradioactive substance and a metal particle.

In certain further embodiments of the above-described methods, theimmunomodulatory peptide is selected from IgX, a fragment of IgX, and avariant of IgX. In certain embodiments the immunomodulatory polypeptidecomprises no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,19, 18, 17, 16, 15, 14, 13 or 12 amino acids. In certain embodiments theimmunomodulatory polypeptide comprises a scFv of an immunoglobulin thatis selected from (i) IgX and (ii) an immunoglobulin that comprises animmunoglobulin polypeptide that is selected from the group consisting ofa polypeptide comprising the amino acid sequence set forth in any one ofSEQ ID NOS:3 and 5-104. In certain embodiments the immunomodulatorypolypeptide comprises a Fab of an immunoglobulin that is selected from(i) IgX and (ii) an immunoglobulin that comprises an immunoglobulinpolypeptide that is selected from the group consisting of a polypeptidecomprising the amino acid sequence set forth in any one of SEQ ID NOS:3and 5-104. In certain embodiments the immunomodulatory polypeptidecomprises a (Fab′)₂ of an immunoglobulin that is selected from (i) IgXand (ii) an immunoglobulin that comprises an immunoglobulin polypeptidethat is selected from the group consisting of a polypeptide comprisingthe amino acid sequence set forth in any one of SEQ ID NOS:3 and 5-104.

In certain embodiments the immunomodulatory polypeptide comprises anintact immunoglobulin heavy chain variable region having an amino acidsequence of an immunoglobulin variable region that is present in animmunoglobulin polypeptide that is selected from the group consisting ofa polypeptide comprising the amino acid sequence set forth in any one ofSEQ ID NOS:3 and 5-104. In certain embodiments the immunomodulatorypolypeptide comprises an intact immunoglobulin heavy chain having anamino acid sequence of an immunoglobulin heavy chain that is selectedfrom the sequences set forth in SEQ ID NOs:3 and 5-104. In certainembodiments the immunomodulatory polypeptide comprises an intactantibody, wherein the antibody comprises an intact immunoglobulin heavychain having an amino acid sequence of an immunoglobulin heavy chainthat is selected from the sequences set forth in SEQ ID NOs: 3 and5-104.

According to certain embodiments there is provided a method of treatinga malignant condition, comprising administering to a subject having orsuspected of having a malignancy at least one composition that isselected from (a) a composition that comprises a therapeuticallyeffective amount of an immunomodulatory polypeptide that compriseseither the amino acid sequence set forth in SEQ ID NO:2 or the aminoacid sequence set forth in SEQ ID NO:106, and (b) a composition thatcomprises a therapeutically effective amount of an antibody, orantigen-binding fragment thereof, that specifically binds to animmunomodulatory polypeptide of no more than 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 amino acids, saidimmunomodulatory polypeptide comprising either the amino acid sequenceset forth in SEQ ID NO:2 or the amino acid sequence set forth in SEQ IDNO:106, and thereby treating the malignant condition. Preferably,treating the malignant condition comprises at least one of killing atumor cell and inhibiting metastasis. In certain embodiments themalignant condition is selected from breast cancer, ovarian cancer,adenoma, colorectal carcinoma, gastric carcinoma, lung carcinoma,prostate carcinoma, hepatocellular carcinoma, melanoma, leukemia andlymphoma.

Certain other embodiments provide a method of treating an autoimmunedisease, comprising administering to a subject having or suspected ofhaving an autoimmune disease a composition that comprises atherapeutically effective amount of an immunomodulatory polypeptide thatcomprises either the amino acid sequence set forth in SEQ ID NO:2 or theamino acid sequence set forth in SEQ ID NO:106, or by administeringantibodies directed against the SEQ ID NO:2 or against the amino acidsequence set forth in SEQ ID NO:106 and thereby treating the autoimmunedisease. In certain further embodiments the autoimmune disease isselected from rheumatoid arthritis, psoriatic arthritis, ulcerativecolitis, Crohn's disease, seronegative spondyloarthopathies, systemiclupus erythematosus, Behcet's disease and vasculitis.

In certain embodiments, there is provided an immunomodulatorypolypeptide that competes with PeptideX2 or a variant thereof forspecific binding to a human neutrophil, wherein said PeptideX2 comprisesthe amino acid sequence set forth in SEQ ID NO:2 and wherein the variantthereof comprises the amino acid sequence set forth in SEQ ID NO:106.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a bar graph that shows IL-4, IL-10 and IL-12 produced by wholeperipheral blood cells from Donor #66 in response to culture with 0,0.1, 1 or 10 ug/ml IgX derived from placental sample BC.

FIG. 2 is a bar graph that shows TNFα and IL-6 produced by wholeperipheral blood cells from Donor #66 in response to culture with 0,0.1, 1 or 10 ug/ml IgX derived from placental sample BC.

FIG. 3 is a bar graph that shows IL-6 produced by whole peripheral bloodcells from Donor #67 in response to culture with 0, 10 or 100 ug/ml IgXderived from placental sample BC.

FIG. 4 is a bar graph that shows IL-2, IL-4, IL-10 and IL-12 produced bywhole peripheral blood cells from Donor #85 in response to culture with0, 10, 100 or 200 ug/ml IgX derived from placental sample BC.

FIG. 5 is a bar graph that shows TNFα produced by whole peripheral bloodcells from Donor #85 in response to culture with 0, 10, 100 or 200 ug/mlIgX derived from placental sample BC.

FIG. 6 is a bar graph that shows IL-6 produced by whole peripheral bloodcells from Donor #85 in response to culture with 0, 10, 100 or 200 ug/mlIgX derived from placental sample BC.

FIG. 7 is a bar graph that shows IL-6 produced by peripheral blood cellsfrom Donor #67 in response to culture with PeptideX1 or PeptideX2.

FIG. 8 is a bar graph that shows IL-2, IL-4, IL-10 and IL-12 produced byperipheral blood cells from Donor #85 in response to culture with 0, 10,100 or 200 ug/ml PeptideX2.

FIG. 9 is a bar graph that shows TNFα and IL-6 produced by peripheralblood cells from Donor #85 in response to culture with 0, 10, 100 or 200ug/ml PeptideX2

FIG. 10 is a bar graph that shows IL-6 produced by peripheral bloodcells in response to culture with 100 or 200 ug/ml of KLH orPeptideX2-KLH conjugate.

FIG. 11 is a bar graph that shows CFUs of E. coli incubated with serum,peripheral blood leukocytes or peripheral blood leukocytes incombination with PeptideX2 for 10 hours.

FIG. 12 is a bar graph that shows CFUs of E. coli incubated with serum,peripheral blood leukocytes or peripheral blood leukocytes incombination with various concentrations of PeptideX2 at several timepoints.

FIG. 13 shows the effects of PeptideX2 (200 μg/mL) on TLR-driven NF-κBactivation in HEK293 cell lines transfected with human TLR2, 3, 4, 5, 7,8 or 9 (InvivoGen, San Diego, Calif.). HEK293 cells (50,000 to 75,000cells/well) expressing the indicated TLR were plated in wells of a96-well plate (200 μl total volume) containing either with no additions,PeptideX2 (in 20 μl buffer), or buffer only (20 μl) as a vehiclecontrol. All cells contained a reporter construct having coding sequencefor Secreted Embryonic Alkaline Phosphatase (SEAP) under the control ofa promoter inducible by the transcription of NF-kB, and were incubatedin media containing a detectable SEAP expression indicator (InvivoGen,San Diego, Calif.). After 16-20 hours incubation at 37° C., the opticaldensity of culture supernatants was read at 650 nm on a MolecularDevices SpectraMax™ 340PC absorbance detector.

FIG. 14 shows the effects of PeptideX2 (200 μg/mL) on TLR-driven NF-κBactivation in HEK293 cell lines transfected with human TLR2, 3, 4, 5, 7,8 or 9 (InvivoGen, San Diego, Calif.), compared to the effects of knownPAMP ligands as agonists for each of these TLRs, as follows: TLR2, heatkilled Listeria monocytogenes (HKLM) at 10⁸ cells/ml; TLR3, poly(I:C) at1 ug/ml; TLR4, E. coli K12 LPS at 100 ng/ml; TLR5, S. typhimuriumflagellin at 100 ng/ml; TLR7, CL097 at 1 ug/ml; TLR8, CL075 at 1 ug/ml;TLR9, CpG ODN 2006 at 100 ng/ml. NF-κB-negative control cell lines werealso tested and produced negative results (not shown).

FIG. 15 shows TLR2-driven NF-κB activation by human TLR2-transfectedHEK293 cells in response to varying concentrations of PeptideX2 in theNF-κB-driven SEAP reporter assay (InvivoGen, San Diego, Calif.).

FIG. 16 shows S. aureus growth in the absence and presence of PeptideX2at 0, 1.5 and 3 hours.

FIG. 17 shows levels of TNFα production by human peripheral white bloodcells (WBC) during short-term co-culture with S. aureus in the presenceand absence of PeptideX2.

FIG. 18 shows the effects of PeptideX2 (200 μg/mL) on TLR-driven NF-κBactivation in HEK293 cell lines transfected with murine TLR2, 3, 4, 5,7, 8 or 9 (InvivoGen, San Diego, Calif.). HEK293 cells (50,000 to 75,000cells/well) expressing the indicated TLR were plated in wells of a96-well plate (200 μl total volume) containing either with no additions,PeptideX2 (in 20 μl buffer), or buffer only (20 μl) as a vehiclecontrol. All cells contained a reporter construct having coding sequencefor Secreted Embryonic Alkaline Phosphatase (SEAP) under the control ofa promoter inducible by the transcription of NF-kB, and were incubatedin media containing a detectable SEAP expression indicator (InvivoGen,San Diego, Calif.). After 16-20 hours incubation at 37° C., the opticaldensity of culture supernatants was read at 650 nm on a MolecularDevices SpectraMax™ 340PC absorbance detector.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the amino acid sequence AEDTAVYYCAR of PeptideX1.

SEQ ID NO:2 is the amino acid sequence KSIAYLQMNSLK of PeptideX2.

SEQ ID NO:3 is the amino acid sequence of AAH90938.1.

SEQ ID NO:4 is the amino acid sequence KSIAYLQMNSLKTEDTALYYCTR,corresponding to amino acid residues at positions 97-119 of AAH90938.1.

SEQ ID NOs:5-104 correspond to the amino acid sequences of Ig heavychains and Ig heavy chain variable regions identified in the BLASTsearch as set forth in Table 2 below.

SEQ ID NO:105 is the amino acid sequence KSIAYLQMNSLKTEDTALYYC,corresponding to amino acid residues at positions 97-117 of AAH90938.1.

SEQ ID NO:106 is the amino acid sequence of general formulaK-X1-X2-X3-YLQM-X4-X5-LK wherein X1 is selected from S and N, X2 isselected from I, T, S, M, R and N, X3 is selected from A, L, V and Q, X4is selected from N, D, S, T and A, and X5 is selected from S, T and N.

SEQ ID NO: 107 is the illustrative spacer amino acid sequenceGlu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp.

SEQ ID NO: 108 is the illustrative spacer amino acid sequenceLys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp.

SEQ ID NO: 109 is the flexible polylinker amino acid sequenceGly-Gly-Gly-Gly-Ser.

SEQ ID NO: 110 is the AAH90938.1 amino acid sequence EVQLVE.

SEQ ID NO: 111 is the amino acid sequence DVQLLE, corresponding to theN-terminal sequence for sample BC H chain.

SEQ ID NOS: 112-115 are IgX H chain partial sequences from fourdifferent placental samples listed in Table 1.

DETAILED DESCRIPTION

The present disclosure relates to immunomodulatory polypeptides andpeptides, and in particular to an unusual placentally derived humanimmunoglobulin described herein and designated IgX, and its derivativepolypeptide PeptideX2 [SEQ ID NO:2] as described herein, includingvariants thereof, which peptides exhibit certain of the immunomodulatoryproperties of IgX. Embodiments of the present invention are based inpertinent part on the discovery of a restricted immunoglobulin (Ig)heavy chain polypeptide, referred to herein as “IgX,” and its surprisingimmunomodulatory effects. Unexpectedly, an immunoglobulin comprising theIgX polypeptide was isolated from human placentas and found to elicitthe expression of cytokines, including in particular interleukin-6(IL-6) and tumor necrosis factor-alpha (TNFα) and also interleukin-10(IL-10), by peripheral blood leukocytes (PBL). This property,qualitatively shared by IgX and PeptideX2 as described herein, is quiteremarkable in that this cytokine expression pattern by PBL is notcharacteristic of either a classic helper T cell type 1 (Th1) or ahelper T cell type 2 (Th2) response. Even more remarkably, an isolateddodecapeptide derived from the heavy chain hypervariable region of IgX,referred to herein as “PeptideX2” [SEQ ID NO:2], exhibits certainimmunomodulatory properties of IgX. Furthermore, and as described ingreater detail herein, PeptideX2 preferentially binds to and thusidentifies a neutrophil subpopulation.

As described herein for the first time and presented in greater detailbelow, the presently provided PeptideX2 directly bound to asubpopulation of neutrophils and also, after being contacted withneutrophil-containing peripheral blood leukocyte preparations, induced,inter alia, elaboration of IL-6 and IL-10. PeptideX2 is also shown herefor the first time to be capable of delivering a specifictranscriptional activation signal to innate immune system cells, viapreferential binding interactions with human or murine TLR2 and/or TLR4(but not other human or murine TLRs) at discrete and detectablesignaling levels that were nevertheless well below the levels deliveredby natural PAMP ligands for these TLRs. In an in vitro sepsis modelsystem that considered early events in white blood cell (e.g., innateimmune cell) interactions with a bacterial pathogen, competitive bindingby PeptideX2 to TLR2 and TLR4 moderated the levels of bacterialPAMP-induced release of the pro-inflammatory cytokine TNFα by peripheralblood white cells, without compromising the ability of these innateimmune system cells to inhibit bacterial (S. aureus) growth. Engagementof neutrophil surface TLR2 and TLR4 by PeptideX2 thus (according tonon-limiting theory) elicited substantially reduced inflammatory indiciarelative to those induced by pathogenic PAMPs for TLR2/TLR4 but did notcompromise neutrophil phagocytic capability, as evidenced by thesecells' ability to kill pathogens.

By these effects on local and/or systemic immunologic status (e.g.,hyperinflammatory vs. hypoinflammatory, altered cytokine profile, etc.),which may vary as a function of PeptideX2 dosage parameters (e.g.,concentration, timing, absence or presence of competing TLR2/4 ligandssuch as PAMPs or DAMPs, activation status of target cells, host clinicalstatus, valency, etc.), the herein described PeptideX2 polypeptides arethus believed according to non-limiting theory to provideunprecedentedly useful immunomodulatory properties. The presentlydisclosed PeptideX2 and IgX polypeptides afford such properties throughtheir ability to alter (e.g., increase or decrease in a statisticallysignificant manner) the activity levels of one or more cellularregulators of immune status, such as biological signals that aretransduced through TLR2 and TLR4.

According to certain preferred embodiments and further according tonon-limiting theory, beneficial uses of the presently disclosedPeptideX2 immunomodulatory polypeptides relate to unexpected advantagesthat are obtained by their dual functioning (i) as weak agonists of TLR2and/or TLR4, through which the biological signals that are delivered byPeptideX2 are transduced but are qualitatively and quantitatively lessprofound than TLR2/TLR4 activation signals that are transduced inresponse to previously described PAMPs, DAMPS or other TLR ligands, and(ii) as antagonists of PAMPs and/or DAMPs by virtue of theircompetitive, albeit low affinity, binding to TLR2 and TLR4. In thisrespect, the presently disclosed PeptideX2 and IgX immunomodulatorypolypeptides surprisingly permit the innate immune system to mediate amoderate inflammatory response instead of the exuberanthyperinflammatory reaction that characterizes early sepsis, withoutdriving the immune system to the immunosuppressed state that otherwiseoften subsequently predominates in later stages of sepsis.

Thus according to certain embodiments disclosed herein there is providedan immunomodulatory polypeptide (e.g., PeptideX2) that, unlike anypreviously described immunologically active agent, is capable of weaklyactivating a human or murine neutrophil subset via TLR2 and/or TLR4 in amanner that rapidly alters the inflammatory cytokine expression profileof such innate immune cells. PeptideX2 surprisingly mediates theseeffects without compromising early-phase antimicrobial activity of thesecells and without activating or inducing the immunosuppressive cytokineprofile that may otherwise be elaborated by phagocytes (macrophages anddendritic cells) involved in the clearance of neutrophils that haveundergone apoptosis or other DAMP.

Accordingly, and without wishing to be bound by theory, it is believedthat the dual weak TLR agonist/potent PAMP/DAMP antagonist activity ofPeptideX2 advantageously avoids permissive conditions for anoverwhelming microbial (e.g., bacterial) infection in sepsis, at leastin part by interfering with the onset of immunosuppressive conditionsthat would otherwise preclude the adaptive immune system from mounting avigorous anti-microbial response against the pathogen. The presentlydisclosed immunomodulatory polypeptides feature the advantageous andpreviously unpredicted properties of (i) binding recognition of, andbeneficially weak signal transduction activity via, human TLR2 and TLR4;(ii) modulation of both TLR2 and TLR4 activation by down-regulating theinnate immune response proinflammatory component when present at lowconcentrations, whilst up-regulating proinflammatory mechanisms athigher concentrations; and (iii) ease of manufacture of an active moietythat may be as small as a dodecapeptide that is expected to benon-immunogenic in humans, avoiding the challenges of monoclonalantibody humanization, CDR optimization and production.

Also without wishing to be bound by theory, it is hypothesized that theherein described placentally derived protein, IgX, contributesmechanistically to allograft success of the fetus in pregnancy and thatthe structural features of IgX which underlie this functional attributereside at least in part in PeptideX2, which forms a portion of the IgXheavy-chain variable (Vh) region and, as disclosed herein, has also beenidentified in the Vh regions of 100 human IgG1(K) immunoglobulins.Further according to non-limiting theory, the immunomodulatorymechanisms that are recruited by IgX, including via PeptideX2 (whichforms a portion of the IgX Vh structure), in the course of protectingthe fetal-maternal allograft from immunological rejection withoutseverely compromising overall immune potential and without triggering amassive inflammatory reaction, may also be usefully exploited in anumber of medically relevant contexts, including improving outcomes inorgan transplantation by decreasing (e.g., in a statisticallysignificant manner relative to appropriate control conditions)immunological rejection mechanisms that may otherwise be manifest inhost responses to organ or tissue grafts, including allografts andxenografts.

The distinctive immunomodulatory properties of IgX and PeptideX2 mayalso find beneficial uses in treating cancer, including promotion ofmechanisms of tumor cell killing and/or in inhibition of metastasis, andalso in treating GVHD in bone marrow transplant recipients, and in thecontext of reproductive medicine, for example, to improve fertility(including pregnancy initiated following in vitro fertilization (IVF))by promoting embryonic implantation, and in reducing the severity ofsymptoms associated with preeclampsia and eclampsia during pregnancy.IgX or PeptideX2, including other polypeptides that contain one or morecopies of the dodecameric Peptide X2 amino acid sequence as set forth inSEQ ID NO:2 such as the human IgG1(K) immunoglobulins presented in Table2, may additionally be advantageously administered in treatingautoimmune diseases such as rheumatoid arthritis, psoriatic arthritis,Crohn's disease, systemic lupus erythematosus and other autoimmunediseases, and may more generally induce a heretofore unprecedentednon-Th1, non-Th2 immunological response that is characterized byup-regulated (e.g., increased in a statistically significant mannerrelative to an appropriate control) release of at least one of (and incertain preferred embodiments at least two of) IL-6, IL-10 and TNFα, byperipheral white blood cells.

According to a certain embodiment disclosed herein, there is providedPeptideX2, an immunomodulatory polypeptide that recognizes andspecifically binds to a cell surface structure that is present oncertain human and murine neutrophils, and which cognate cell surfacestructure resides in the toll-like receptors TLR2 and TLR4. Theimmunomodulatory polypeptides described herein, for instance, IgX andPeptideX2 and other PeptideX2-containing immunoglobulins such as thosedescribed herein, may thus find immunotherapeutic uses.

For example, in certain embodiments, the presently disclosed PeptideX2immunomodulatory polypeptides, which comprise the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:106, may be beneficially exploited to treat, orto reduce (e.g., decrease with statistical significance) the severity orlikelihood of occurrence of, sepsis in a subject such as a human patientor other mammal. Accordingly and without intending to be bound bytheory, these immunomodulatory polypeptides may thus be useful as agentsthat alter (e.g., increase or decrease in a statistically significantmanner, and in certain preferred embodiments, decrease) PAMP ligandbinding to neutrophil TLR2 and/or TLR4, by binding to TLR2 and/or TLR4without eliciting the neutrophil activation that would otherwisecharacterize a full-fledged TLR-ligand response.

In these and other related embodiments, the presently disclosedimmunomodulatory polypeptides may find uses as agents for regulatedimmunosuppressive treatment. In certain contemplated embodiments, theseand related PeptideX2-containing immunomodulatory polypeptides mayusefully suppress immune effector cell proliferation in response to anallograft or xenograft, without severely compromising host immunepotential and without inducing massive inflammation. The hereindisclosed immunomodulatory polypeptides may, for example by way ofillustration and not limitation, be used in transplant patients toprevent or ameliorate graft rejection and/or GVHD.

In certain other embodiments the invention relates to the use of theherein disclosed immunomodulatory polypeptides (e.g., PeptideX2, IgX, orother PeptideX2-containing polypeptides) in fertility treatments, andparticularly for use in increasing the likelihood of embryonicimplantation in a variety of contexts, including natural pregnancies aswell as those that may result from introduction into a surrogate mother(such as a pseudopregnant female) of an embryo produced by IVF. In otherembodiments, the herein disclosed immunomodulatory polypeptides(PeptideX2, IgX, or other PeptideX2-containing polypeptides) may be usedin the prevention and/or treatment of pregnancy related conditionsincluding preeclampsia and eclampsia. In such applications of the hereindescribed compositions and methods, the present immunomodulatorypolypeptide may be contacted with the embryo in vitro or in vivo and/orwith the pregnant or pseudopregnant female in whom it is desired topromote fertility.

Other herein contemplated embodiments encompass the use of the disclosedimmunomodulatory polypeptides (e.g., PeptideX2, IgX, or otherPeptideX2-containing polypeptides) or the use of engineered antibodiesspecifically targeted to bind to peptideX2, IgX, or otherpeptideX2-containing polypeptides in order to control immunity andinflammation, for example, as controlled immunosuppressants in thetreatment of autoimmune diseases and disorders as provided herein, suchas rheumatoid arthritis, psoriatic arthritis, Crohn's disease, andothers.

As described herein, placentally derived IgX having unusualimmunological properties contains in its heavy chain sequence a regionin the vicinity of the Vh CDR3 the amino acid sequence referred to asPeptideX2:

[SEQ ID NO: 2] KSIAYLQMNSLK

Immunomodulatory properties of IgX are presented below include theability to induce release of IL-6, IL-10 and TNFα by peripheral bloodwhite cells, a property shared qualitatively by PeptideX2. Accordingly,certain useful embodiments as disclosed herein contemplate exploitingthese and related properties of PeptideX2 and/or PeptideX2-containingpolypeptides of no more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13 or 12 amino acids, for example apolypeptide of sequence:

[SEQ ID NO: 105] KSIAYLQMNSLKTEDTALYYC

In certain other useful embodiments the IgX immunomodulatory propertiesreside in structurally related polypeptides, including polypeptides ofno more than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13 or 12 amino acids, that contain the amino acid sequenceof general formula:

K-X1-X2-X3-YLQM-X4-X5-LK as set forth in SEQ ID NO:106,

wherein X1 is selected from S and N, X2 is selected from I, T, S, M, Rand N, X3 is selected from A, L, V and Q, X4 is selected from N, D, S, Tand A, and X5 is selected from S, T and N. In certain such embodimentswherein the polypeptide comprises a sequence having the general formulaof SEQ ID NO:106 but in which the included sequence according to SEQ IDNO:106 is not the same as the sequence set forth in SEQ ID NO:2,polypeptides of more than 31 amino acids are also contemplated, such aspolypeptides of 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61-70,71-80, 81-90, 91-100 or more amino acids.

Certain other embodiments contemplate exploiting these and relatedproperties of larger PeptideX2-containing polypeptides, in particular,immunoglobulin heavy chain variable region polypeptides that contain thePeptideX2 sequence (SEQ ID NO:2) or the sequence set forth in SEQ IDNO:106 as part of their primary structures, for instance, any of thehuman IgG1 heavy chain polypeptides (or Vh regions thereof or fragmentsor variants thereof so long as SEQ ID NO:2 or SEQ ID NO:106 is present)such as the human IgG1(K) heavy chains identified in Table 2.

It will therefore be appreciated that in certain herein disclosedembodiments an isolated immunomodulatory polypeptide comprising thePeptideX2 sequence set forth in SEQ ID NO:2 or SEQ ID NO:106 as providedherein may comprise 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13 or 12 amino acids, with SEQ ID NO:2 or SEQ IDNO:106 being situated at the N-terminus with 0-19 amino acids of anysequence forming the C-terminus (e.g., as is the case in SEQ ID NOS:4and 105), or SEQ ID NO:2 or SEQ ID NO:106 may be situated at theC-terminus with 0-19 amino acids of any sequence forming the N-terminus,or SEQ ID NO:2 or SEQ ID NO:106 may be situated at neither theN-terminus nor the C-terminus and may be linked via peptide bonds toadditional amino acids of any sequence that form N- and C-termini, solong as the entire polypeptide is of no more than 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 or 14 amino acids inlength. As also noted above, longer polypeptides are also contemplatedin which is contained the sequence according to SEQ ID NO:106 so long asthe so-contained sequence differs from SEQ ID NO:2 in at least one ofthe amino acid positions X1, X2, X3, X4 or X5 of SEQ ID NO:106.

According to certain other embodiments the PeptideX2-containingpolypeptide need not be so limited and may comprise larger polypeptidesequences such as the SEQ ID NO:2-containing IgG1 Vh domain polypeptidesequences presented in Tables 1 and 2.

The presently disclosed embodiments for the first time provide specificimmunoglobulin antigen-combining (V) region fragments havingimmunomodulatory properties as described herein, which have notpreviously been recognized. Depressed levels of overallendometrial/decidual IgG levels have previously been correlated withadvancing trimesters during the course of pregnancy (Kutteh et al., 2001Am J Obstet. Gynecol. 184:865) but no identification of individual IgGspecies or their specificities or biological effects have beendescribed. Prior reports of an immunoglobulin component that was presentin immunosuppressive endometrial extracts traced the immunosuppressiveactivity to the immunoglobulin constant (Fc) domain (Haruyama et al.,1991 J. Reprod. Immunol. 19:1; Kitano et al, 1990 Acta Obst. Gynaec.Jpn. 42(7):739), which does not mediate specific antigen recognition andbinding. By pointing to a non-antigen specific biological effectmediated by the immunoglobulin constant (C) region, these earlierreports teach away from the presently described IgX and PeptideX2polypeptides, which comprise immunomodulatory polypeptide regions of adistinctive immunoglobulin variable (V) region sequence.

Polypeptides and Proteins

The terms “polypeptide” “protein” and “peptide” and “glycoprotein” areused interchangeably and mean a polymer of amino acids not limited toany particular length. The term does not exclude modifications such asmyristylation, sulfation, glycosylation, phosphorylation and addition ordeletion of signal sequences. The terms “polypeptide” or “protein” meansone or more chains of amino acids, wherein each chain comprises aminoacids covalently linked by peptide bonds, and wherein said polypeptideor protein can comprise a plurality of chains non-covalently and/orcovalently linked together by peptide bonds, having the sequence ofnative proteins, that is, proteins produced by naturally-occurring andspecifically non-recombinant cells, or genetically-engineered orrecombinant cells, and comprise molecules having the amino acid sequenceof the native protein, or molecules having deletions from, additions to,and/or substitutions of one or more amino acids of the native sequence.Thus, a “polypeptide” or a “protein” can comprise one (termed “amonomer”) or a plurality (termed “a multimer”) of amino acid chains. Theterms “peptide,” “polypeptide” and “protein” specifically encompass theimmunomodulatory polypeptides of the present disclosure, or sequencesthat have deletions from, additions to, and/or substitutions of one ormore amino acid of an immunomodulatory polypeptide.

The terms “isolated protein” and “isolated polypeptide” referred toherein means that a subject protein or polypeptide (1) is free of atleast some other proteins or polypeptides with which it would typicallybe found in nature, (2) is essentially free of other proteins orpolypeptides from the same source, e.g., from the same species, (3) isexpressed by a cell from a different species, (4) has been separatedfrom at least about 50 percent of polynucleotides, lipids,carbohydrates, or other materials with which it is associated in nature,(5) is not associated (by covalent or noncovalent interaction) withportions of a protein or polypeptide with which the “isolated protein”or “isolated polypeptide” may be associated in nature, (6) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, or (7) does not occur innature. Such an isolated protein or polypeptide can be encoded bygenomic DNA, cDNA, mRNA or other RNA, of may be of synthetic originaccording to any of a number of well known chemistries for artificialpeptide and protein synthesis, or any combination thereof. In certainembodiments, the isolated protein or polypeptide is substantially freefrom proteins or polypeptides or other contaminants that are found inits natural environment that would interfere with its use (therapeutic,diagnostic, prophylactic, research or otherwise).

The term “polypeptide fragment” refers to a polypeptide, which can bemonomeric or multimeric, that has an amino-terminal deletion, acarboxyl-terminal deletion, and/or an internal deletion or substitutionof a naturally-occurring or recombinantly-produced polypeptide. As usedherein, “contiguous amino acids” refers to covalently linked amino acidscorresponding to an uninterrupted linear portion of a disclosed aminoacid sequence. In certain embodiments, a polypeptide fragment cancomprise an amino acid chain at least 5 to about 500 amino acids long.It will be appreciated that in certain embodiments, fragments are atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long.

Polypeptides may comprise a signal (or leader) sequence at theN-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be fused in-frame or conjugated to a linker or other sequencefor ease of synthesis, purification or identification of the polypeptide(e.g., poly-His), or to enhance binding of the polypeptide to a solidsupport. Fusion domain polypeptides may be joined to the polypeptide atthe N-terminus and/or at the C-terminus, and may include as non-limitingexamples, immunoglobulin-derived sequences such as Ig constant regionsequences or portions thereof, affinity tags such as His tag (e.g.,hexahistidine or other polyhistidine), FLAG™ or myc or other peptideaffinity tags, detectable polypeptide moieties such as green fluorescentprotein (GFP) or variants thereof (e.g., yellow fluorescent protein(YFP), blue fluorescent protein (BFP), other aequorins or derivativesthereof, etc.) or other detectable polypeptide fusion domains, enzymesor portions thereof such as glutathione-S-transferase (GST) or otherknown enzymatic detection and/or reporter fusion domains, and the like,as will be familiar to the skilled artisan.

Cysteine-containing peptides may be used as fusion peptides that can bejoined to the N- and/or C-terminus of the herein described peptide X2(e.g., SEQ ID NO:2) or peptide X2-like (e.g., SEQ ID NO:106) containingpolypeptides to permit ready assembly of such polypeptides intodisulfide-crosslinked dimers, trimers, tetramers or higher multimersaccording to established methodologies. For example, fusion polypeptidescontaining immunoglobulin gene superfamily member-derived sequences thatinclude cysteine residues capable of forming interchain disulfidebridges are well known, as also are other strategies for engineering S-Slinked multimers (e.g., Reiter et al., 1994 Prot. Eng. 7:697; Zhu etal., 1997 Prot. Sci. 6:781; Mabry et al., 2010 Mabs 2:20; Gao et al.,1999 Proc. Nat. Acad. Sci. USA 96:6025; Lim et al., 2010 Biotechnol.Bioeng. 106:27) Alternative approaches are also contemplated forgrafting peptide sequences that promote multimer assembly as fusiondomains onto a desired polypeptide such as the herein describedimmunomodulatory peptides (e.g., Fan et al., 2008 FASEB J. 22:3795).

Polypeptide modifications may be effected biosynthetically and/orchemically according to a wide variety of well known methodologies, andmay also include conjugation to carrier proteins (e.g., keyhole limpethemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA) or othermolecules), and covalent or non-covalent immobilization on solidsupports. Chemical or biosynthetic conjugation to a carrier iscontemplated, according to certain embodiments, for generation ofconjugates that are multivalent with respect to the herein describedpeptide X2 (e.g., a polypeptide that contains SEQ ID NO:2) or peptideX2-like (e.g., a polypeptide that contains SEQ ID NO:106) moieties. Forexample and according to non-limiting theory, it is believed that targetcells for immunomodulation (e.g., a neutrophil subset) that expresscertain cell surface receptors for peptide X2 may transduce biologicalsignals more efficiently when stimulated by a multivalent peptide X2structure, relative to the level of induction that may be afforded by amonovalent peptide X2 structure. Also contemplated is detectablelabeling with detectable indicator moieties (sometimes referred to asreporter moieties) such as fluorophores (e.g., FITC, TRITC, Texas Red,etc.). Examples of a broad range of detectable indicators (includingcolorimetric indicators) that may be selected for specific purposes aredescribed in Haugland, 2002 Handbook of Fluorescent Probes and ResearchProducts—Ninth Ed., Molecular Probes, Eugene, Oreg.; in Mohr, 1999 J.Mater. Chem., 9: 2259-2264; in Suslick et al., 2004 Tetrahedron60:11133-11138; and in U.S. Pat. No. 6,323,039. (See also, e.g., FlukaLaboratory Products Catalog, 2001 Fluka, Milwaukee, Wis.; and Sigma LifeSciences Research Catalog, 2000, Sigma, St. Louis, Mo.) A detectableindicator may be a fluorescent indicator, a luminescent indicator, aphosphorescent indicator, a radiometric indicator, a dye, an enzyme, asubstrate of an enzyme, an energy transfer molecule, or an affinitylabel.

Other detectable indicators for use in certain embodiments contemplatedherein include affinity reagents such as antibodies, lectins,immunoglobulin Fc receptor proteins (e.g., Staphylococcus aureus proteinA, protein G or other Fc receptors), avidin, biotin, other ligands,receptors or counterreceptors or their analogues or mimetics, and thelike. For such affinity methodologies, reagents for immunometricmeasurements, such as suitably labeled antibodies or lectins, may beprepared including, for example, those labeled with radionuclides, withfluorophores, with affinity tags, with biotin or biotin mimeticsequences or those prepared as antibody-enzyme conjugates (see, e.g.,Weir, D. M., Handbook of Experimental Immunology, 1986, BlackwellScientific, Boston; Scouten, W. H., 1987 Methods in Enzymology135:30-65; Harlow and Lane, Antibodies: A Laboratory Manual, 1988 ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Haugland, Handbookof Fluorescent Probes and Research Products—Ninth Ed., 2002 MolecularProbes, Eugene, Oreg.; Scopes, R. K., Protein Purification: Principlesand Practice, 1987, Springer-Verlag, NY; Hermanson, G. T. et al.,Immobilized Affinity Ligand Techniques, 1992, Academic Press, Inc., NY;Luo et al., 1998 J. Biotechnol. 65:225 and references cited therein).

A peptide linker/spacer sequence may also be employed to separatemultiple polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and/or tertiary structures, ifdesired. Such a peptide linker sequence can be incorporated into afusion polypeptide using standard techniques well known in the art.

Certain peptide spacer sequences may be chosen, for example, based on:(1) their ability to adopt a flexible extended conformation; (2) theirinability to adopt a secondary structure that could interact withfunctional epitopes on the first and second polypeptides; and/or (3) thelack of hydrophobic or charged residues that might react with thepolypeptide functional epitopes.

In one illustrative embodiment, peptide spacer sequences contain, forexample, Gly, Asn and Ser residues. Other near neutral amino acids, suchas Thr and Ala, may also be included in the spacer sequence.

Other amino acid sequences which may be usefully employed as spacersinclude those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphyet al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. Nos.4,935,233 and 4,751,180.

Other illustrative spacers may include, for example,Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO: 107)(Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1066-1070) andLys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp(SEQ ID NO: 108) (Bird et al., 1988, Science 242:423-426).

In some embodiments, spacer sequences are not required when the firstand second polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference. Two coding sequences can be fused directly without anyspacer or by using a flexible polylinker composed, for example, of thepentamer Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 109) repeated 1 to 3 times.

In certain illustrative embodiments, a peptide spacer is between 1 toamino acids, between 5 to 10 amino acids, between 5 to 25 amino acids,between 5 to 50 amino acids, between 10 to 25 amino acids, between 10 to50 amino acids, between 10 to 100 amino acids, or any intervening rangeof amino acids.

In other illustrative embodiments, a peptide spacer comprises about 1,5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids in length.

Amino acid sequence modification(s) of the immunomodulatory polypeptides(e.g., PeptideX2 [SEQ ID NO:2] sequence-containing polypeptides)described herein are contemplated, including polypeptides that containin their sequence the amino acid sequence set forth in SEQ ID NO:106.For example, it may be desirable to improve the binding affinity and/orother biological properties of the immunomodulatory polypeptide. Forexample, amino acid sequence variants may be prepared by introducingappropriate nucleotide changes into a polynucleotide that encodes theimmunomodulatory polypeptide or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequence of theimmunomodulatory polypeptide. Any combination of deletion, insertion,and substitution may be made to arrive at the final immunomodulatorypolypeptide variant, provided that the final construct possesses thedesired characteristics (e.g., suppresses cell proliferation in a mixedlymphocyte reaction, or induces elaboration by peripheral blood whitecells of the non-Th1/non-Th2 cytokine profile as described herein, orexhibits activity in a preimplantation factor activity assay such asthose described in U.S. Pat. Nos. 5,646,003, 5,981,198, or WO2005/040196). The amino acid changes also may alter post-translationalprocesses of the immunomodulatory polypeptide, such as changing thenumber or position of glycosylation sites.

Determination of the three-dimensional structures of representativepolypeptides (e.g., PeptideX2 or a SEQ ID NO:2-containing polypeptide)may be made through routine methodologies such that substitution,addition, deletion or insertion of one or more amino acids with selectednatural or non-natural amino acids can be virtually modeled for purposesof determining whether a so derived structural variant retains thespace-filling properties of presently disclosed species. See, forinstance, Donate et al., 1994 Prot. Sci. 3:2378; Bradley et al., Science309: 1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005);Dietz et al., Proc. Nat. Acad. Sci. USA 103:1244 (2006); Dodson et al.,Nature 450:176 (2007); Qian et al., Nature 450:259 (2007); Raman et al.Science 327:1014-1018 (2010). Some additional non-limiting examples ofcomputer algorithms that may be used for these and related embodiments,such as for rational design of immunomodulatory polypeptides as providedherein, include VMD which is a molecular visualization program fordisplaying, animating, and analyzing large biomolecular systems using3-D graphics and built-in scripting (see the website for the Theoreticaland Computational Biophysics Group, University of Illinois atUrbana-Champagne, at ks.uiuc.edu/Research/vmd/.

Many other computer programs are known in the art and available to theskilled person and which allow for determining atomic dimensions fromspace-filling models (van der Waals radii) of energy-minimizedconformations; GRID, which seeks to determine regions of high affinityfor different chemical groups, thereby enhancing binding, Monte Carlosearches, which calculate mathematical alignment, and CHARMM (Brooks etal. (1983) J. Comput. Chem. 4:187-217) and AMBER (Weiner et al (1981) J.Comput. Chem. 106: 765), which assess force field calculations, andanalysis (see also, Eisenfield et al. (1991) Am. J. Physiol.261:C376-386; Lybrand (1991) J. Pharm. Belg. 46:49-54; Froimowitz (1990)Biotechniques 8:640-644; Burbam et al. (1990) Proteins 7:99-111;Pedersen (1985) Environ. Health Perspect. 61:185-190; and Kini et al.(1991) J. Biomol. Struct. Dyn. 9:475-488). A variety of appropriatecomputational computer programs are also commercially available, such asfrom Schrödinger (Munich, Germany).

Antibodies

Certain embodiments of the present invention include antibodies thatspecifically bind to a PeptideX2 [SEQ ID NO:2]-containing polypeptide asprovided herein, while certain other embodiments include antibodies thatthemselves include the PeptideX2 sequence set forth in SEQ ID NO:2. Asdescribed herein, PeptideX2 (SEQ ID NO:2) was identified as a region ofplacentally derived antibody IgX, and PeptideX2 shares with IgX certainadvantageous and unexpected immunomodulatory properties.

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity, e.g., specifically bind to neutrophils. The term“immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

The basic antibody unit is a heterotetrameric glycoprotein composed oftwo identical light (L) chains and two identical heavy (H) chains. EachL chain is linked to an H chain by one covalent disulfide bond, whilethe two H chains are linked to each other by one or more disulfide bondsdepending on the H chain isotype. Each H and L chain also has regularlyspaced intrachain disulfide bridges. Each H chain has at the N-terminusa variable domain (V_(H)) followed by three constant domains (C_(H)) foreach of the α and γ chains and four C_(H) domains for μ and ε isotypes.Each L chain has at the N-terminus, a variable domain (V_(L)) followedby a constant domain (CL) at its other end. The V_(L) is aligned withthe V_(H) and the CL is aligned with the first constant domain of theheavy chain (C_(H)1). Particular amino acid residues are believed toform an interface between the light chain and heavy chain variabledomains. The pairing of a V_(H) and V_(L) together forms a singleantigen-binding site.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa (κ) and lambda (λ), based on theamino acid sequences of their constant domains (C_(L)). Depending on theamino acid sequence of the constant domain of their heavy chains(C_(H)), immunoglobulins can be assigned to different classes orisotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG,and IgM, having heavy chains designated alpha (α), delta (δ), epsilon(ε), gamma (γ) and mu (μ), respectively. The γ and a classes are furtherdivided into subclasses on the basis of relatively minor differences inC_(H) sequence and function, e.g., humans express the followingsubclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. It will beappreciated that mammals encoding multiple Ig isotypes will be able toundergo isotype class switching.

An IgM antibody consists of 5 of the basic heterotetramer units alongwith an additional polypeptide called J chain, and therefore contains 10antigen binding sites, while secreted IgA antibodies can polymerize toform polyvalent assemblages comprising 2-5 of the basic 4-chain unitsalong with J chain. In the case of IgG, the 4-chain unit is generallyabout 150,000 daltons. For the structure and properties of the differentclasses of antibodies, see, e.g., Basic and Clinical Immunology, 8thedition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.),Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.

The V domain mediates antigen binding and defines specificity of aparticular antibody for its particular antigen. The gene sequenceencoding the V_(H) domain has multiple copies of variable (V), diversity(D), and joining (J) segments. The gene sequence encoding the V_(L)domain contains multiple copies of V and J segments. The V_(H) and V_(L)regions undergo gene rearrangement (i.e., somatic recombination) todevelop diverse antigen specificity in antibodies. The term “variable”refers to the fact that certain segments of the V domains differextensively in sequence among antibodies.

However, the variability is not evenly distributed across the 110-aminoacid span of the variable domains. Instead, the V regions consist ofrelatively invariant stretches called framework regions (FRs) of 15-30amino acids separated by short regions of extreme variability called“hypervariable regions.” These hypervariable regions are the result ofsomatic hypermutation during the affinity maturation process, and theyare typically each 9-18 amino acids long. However, they have been foundto range from 4-28 amino acids in length depending upon the particularepitope. For example, CDR3 regions up to at least 22 or 23 amino acidsin length have been described. See, e.g., Morea V, et al., J Mol Biol.275(2):269-94 (1998) and Kabat, E. A., et al., Sequences of Proteins ofImmunological Interest, Fifth Edition. NIH Publication No. 91-3242(1991).

The variable domains of native heavy and light chains each comprise fourFRs, largely adopting a β-sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming part of, the β-sheet structure. The hypervariable regions ineach chain are held together in close proximity by the FRs and, with thehypervariable regions from the other chain, contribute to the formationof the antigen-binding site of antibodies (see Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 28-36(H1), 50-65 (H2) and 95-102 (H3) in the V_(H); Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g., residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the V_(L), and 26-32 (H1), 53-55 (H2) and96-101 (H3) in the V_(H); Chothia and Lesk, J. Mol. Biol. 196:901-917(1987)).

An “isolated antibody” is one that has been separated and/or recoveredfrom a component of its natural environment. Contaminant components ofits natural environment are materials that would interfere withdiagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In preferred embodiments, the antibody is purified: (1) to greater than95% by weight of antibody as determined by the Bradford method, and mostpreferably more than 99% by weight; (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGEunder reducing or non-reducing conditions using Coomassie blue or,silver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

An “intact” antibody is one that comprises an antigen-binding site aswell as a CL and at least heavy chain constant domains, C_(H) 1, C_(H) 2and C_(H) 3. The constant domains may be native sequence constantdomains (e.g., human native sequence constant domains) or amino acidsequence variants thereof. Preferably, the intact antibody has one ormore effector functions.

An “antibody fragment” is a polypeptide comprising or consisting of aportion of an intact antibody, preferably the antigen binding orvariable region of the intact antibody. Examples of antibody fragmentsinclude Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linearantibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H) 1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment thatroughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Both the Fab and F(ab′)₂ are examples of “antigen-bindingfragments.” Fab′ fragments differ from Fab fragments by havingadditional few residues at the carboxy terminus of the C_(H)1 domainincluding one or more cysteines from the antibody hinge region. Fab′-SHis the designation herein for Fab′ in which the cysteine residue(s) ofthe constant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments that have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “Fc” fragment comprises the carboxy-terminal portions (i.e., the CH2and CH3 domains) of both H chains held together by disulfides. Theeffector functions of antibodies are determined by sequences in the Fcregion. The Fc domain is the portion of the antibody recognized by cellreceptors, such as the FcR, and to which the complement-activatingprotein, C1q, binds.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and antigen-binding site. This fragment consists ofa dimer of one heavy- and one light-chain variable region domain intight, non-covalent association. From the folding of these two domainsemanate six hypervariable loops (three loops each from the H and Lchain) that contribute the amino acid residues for antigen binding andconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

As used herein, the term “polyclonal antibody” refers to an antibodyobtained from a population of antigen-specific antibodies that recognizemore than one epitope of the specific antigen. “Antigen” or “immunogen”refers to a peptide, lipid, polysaccharide or polynucleotide which isrecognized by the adaptive immune system. Antigens may be self ornon-self molecules. Examples of antigens include, but are not limitedto, bacterial cell wall components, pollen, and rh factor. The region ofan antigen that is specifically recognized by a specific antibody is an“epitope” or “antigenic determinant.” A single antigen may have multipleepitopes.

The term “monoclonal antibody” (mAb) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto polyclonal antibody preparations that include different antibodiesdirected against different epitopes, each monoclonal antibody isdirected against a single epitope of the antigen. In addition to theirspecificity, the monoclonal antibodies are advantageous in that they maybe synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein include “chimeric antibodies” in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see, U.S. Pat. Nos. 4,816,567; 5,530,101 and7,498,415; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)). For example, chimeric antibodies may comprise human andnon-human residues. Furthermore, chimeric antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. For further details, see Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol. 2:593-596 (1992). Chimeric antibodies also includeprimatized and humanized antibodies.

A “humanized antibody” is generally considered to be a human antibodythat has one or more amino acid residues introduced into it from asource that is non-human. These non-human amino acid residues aretypically taken from a variable domain. Humanization is traditionallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting non-human variable sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. Nos. 4,816,567; 5,530,101 and7,498,415) wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In some instances, a “humanized” antibody is onewhich is produced by a non-human cell or animal and comprises humansequences, e.g., He domains.

A “human antibody” is an antibody containing only sequences present inan antibody naturally produced by a human. However, as used herein,human antibodies may comprise residues or modifications not found in anaturally occurring human antibody, including those modifications andvariant sequences described herein. These are typically made to furtherrefine or enhance antibody performance. In some instances, humanantibodies are produced by transgenic animals. For example, see U.S.Pat. Nos. 5,770,429; 6,596,541 and 7,049,426.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

The phrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of ananti-PeptideX2 antibody is one that can bind to PeptideX2.

An antibody having a “biological characteristic” of a designatedantibody is one that possesses one or more of the biologicalcharacteristics of that antibody which distinguish it from otherantibodies. For example, in certain embodiments, an antibody with abiological characteristic of a designated antibody will bind the sameepitope as that bound by the designated antibody and/or have a commoneffector function as the designated antibody.

As used herein, an antibody is said to be “immunospecific,” “specificfor” or to “specifically bind” an antigen if it reacts at a detectablelevel with the antigen, preferably with an affinity constant, K_(a), ofgreater than or equal to about 10⁴ M⁻¹, or greater than or equal toabout 10⁵ M⁻¹, greater than or equal to about 10⁶ M⁻¹, greater than orequal to about 10⁷ M⁻¹, or greater than or equal to 10⁸ M⁻¹. Affinity ofan antibody for its cognate antigen is also commonly expressed as adissociation constant K_(D), and in certain embodiments, aPeptideX2-specific antibody specifically binds to PeptideX2 if it bindswith a K_(D) of less than or equal to 10⁻⁴ M, less than or equal toabout 10⁻⁵ M, less than or equal to about 10⁻⁶ M, less than or equal to10⁻⁷ M, or less than or equal to 10⁻⁸ M. Affinities of antibodies can bereadily determined using conventional techniques, for example, thosedescribed by Scatchard et al. (Ann. N. Y. Acad. Sci. USA 51:660 (1949)).

Binding properties of an antibody to antigens, cells or tissues thereofmay generally be determined and assessed using immunodetection methodsincluding, for example, immunofluorescence-based assays, such asimmuno-histochemistry (IHC) and/or fluorescence-activated cell sorting(FACS).

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as polysorbate 20 (TWEEN™)polyethylene glycol (PEG), and poloxamers (PLURONICS™), and the like.

Nucleic Acids and Polynucleotides

Immunomodulatory polypeptides as provided herein, and encoding nucleicacid molecules and vectors, may be isolated and/or purified, e.g. fromtheir natural environment, in substantially pure or homogeneous form,or, in the case of nucleic acid, free or substantially free of nucleicacid or genes of origin other than the sequence encoding a polypeptidewith the desired function. Nucleic acid may comprise DNA or RNA and maybe wholly or partially synthetic. Reference to a nucleotide sequence asset out herein encompasses a DNA molecule with the specified sequence,and encompasses a RNA molecule with the specified sequence in which U issubstituted for T, unless context requires otherwise.

The present invention thus further provides in certain embodiments anisolated nucleic acid encoding PeptideX2 (comprising the amino acidsequence set forth in SEQ ID NO:2) or a polypeptide of 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 aminoacids that includes the dodecameric sequence of PeptideX2, including theIgX heavy chain and also including, for example, any of the polypeptideshaving the amino acid sequences set forth in SEQ ID NO:3 and SEQ IDNOS:5-104. As described below (see Examples), the PeptideX2 sequence mayoccur within or partially within a defined immunoglobulincomplementarity determining region (CDR) in such sequences, such that incertain embodiments an isolated nucleic acid that comprises apolynucleotide sequence encoding SEQ ID NO:2 may comprise all or aportion of an immunoglobulin chain-encoding polynucleotide (e.g., animmunoglobulin heavy chain such as a human gamma chain).

Certain other embodiments additionally contemplate an antibody orantigen-binding fragment thereof that specifically binds to PeptideX2 asdescribed herein, for instance, an antibody that may itself be animmunomodulatory polypeptide that competes with PeptideX2 for specificbinding to a human neutrophil. Certain related embodiments may thereforecontemplate a nucleic acid which codes for an anti-PeptideX2immunoglobulin complementarity determining region (CDR) or heavy-chainvariable (VH) or light-chain variable (VL) domain as described herein.Nucleic acids include DNA and RNA. These and related embodiments mayinclude polynucleotides encoding immunomodulatory polypeptides asdescribed herein. The term “isolated polynucleotide” as used hereinshall mean a polynucleotide of genomic, cDNA, or synthetic origin orsome combination thereof, wherein by virtue of its origin the isolatedpolynucleotide (1) is not associated with all or a portion of apolynucleotide in which the isolated polynucleotide is found in nature,(2) is linked to a polynucleotide to which it is not linked in nature,or (3) does not occur in nature as part of a larger sequence.

Immunomodulatory polypeptides as provided herein, and encoding nucleicacid molecules and vectors, may be isolated and/or purified, e.g. fromtheir natural environment, in substantially pure or homogeneous form,or, in the case of nucleic acid, free or substantially free of nucleicacid or genes of origin other than the sequence encoding a polypeptidewith the desired function. Nucleic acid may comprise DNA or RNA and maybe wholly or partially synthetic. Reference to a nucleotide sequence asset out herein encompasses a DNA molecule with the specified sequence,and encompasses a RNA molecule with the specified sequence in which U issubstituted for T, unless context requires otherwise.

The term “operably linked” means that the components to which the termis applied are in a relationship that allows them to carry out theirinherent functions under suitable conditions. For example, atranscription control sequence “operably linked” to a protein codingsequence is ligated thereto so that expression of the protein codingsequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

The term “control sequence” as used herein refers to polynucleotidesequences that can affect expression, processing or intracellularlocalization of coding sequences to which they are ligated or operablylinked. The nature of such control sequences may depend upon the hostorganism. In particular embodiments, transcription control sequences forprokaryotes may include a promoter, ribosomal binding site, andtranscription termination sequence. In other particular embodiments,transcription control sequences for eukaryotes may include promoterscomprising one or a plurality of recognition sites for transcriptionfactors, transcription enhancer sequences, transcription terminationsequences and polyadenylation sequences. In certain embodiments,“control sequences” can include leader sequences and/or fusion partnersequences.

The term “polynucleotide” as referred to herein means single-stranded ordouble-stranded nucleic acid polymers. In certain embodiments, thenucleotides comprising the polynucleotide can be ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.Such modifications may include base modifications such as bromouridine,ribose modifications such as arabinoside and 2′,3′-dideoxyribose andinternucleotide linkage modifications such as phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term“polynucleotide” specifically includes single and double stranded formsof DNA.

The term “naturally occurring nucleotides” includes deoxyribonucleotidesand ribonucleotides. The term “modified nucleotides” includesnucleotides with modified or substituted sugar groups and the like. Theterm “oligonucleotide linkages” includes oligonucleotide linkages suchas phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See, e.g., LaPlanche et al., 1986, Nucl.Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077;Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991,Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES ANDANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), OxfordUniversity Press, Oxford England; Stec et al., U.S. Pat. No. 5,151,510;Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures ofwhich are hereby incorporated by reference for any purpose. Anoligonucleotide can include a detectable label to enable detection ofthe oligonucleotide or hybridization thereof.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information to a host cell.The term “expression vector” refers to a vector that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control expression of inserted heterologous nucleic acidsequences. Expression includes, but is not limited to, processes such astranscription, translation, and RNA splicing, if introns are present.

As will be understood by those skilled in the art, polynucleotides mayinclude genomic sequences, extra-genomic and plasmid-encoded sequencesand smaller engineered gene segments that express, or may be adapted toexpress, proteins, polypeptides, peptides and the like. Such segmentsmay be naturally isolated, or modified synthetically by the skilledperson.

As will be also recognized by the skilled artisan, polynucleotides maybe single-stranded (coding or antisense) or double-stranded, and may beDNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules mayinclude HnRNA molecules, which contain introns and correspond to a DNAmolecule in a one-to-one manner, and mRNA molecules, which do notcontain introns.

Additional coding or non-coding sequences may, but need not, be presentwithin a polynucleotide according to the present disclosure, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials. Polynucleotides may comprise a native sequence or maycomprise a sequence that encodes a variant or derivative of such asequence.

Therefore, according to these and related embodiments, the presentdisclosure also provides polynucleotides encoding the immunomodulatorypolypeptides (e.g., PeptideX2, IgX, and other PeptideX2sequence-containing polypeptides) described herein. In certainembodiments, polynucleotides are provided that comprise some or all of apolynucleotide sequence encoding a peptide as described herein andcomplements of such polynucleotides.

In other related embodiments, polynucleotide variants may havesubstantial identity to a polynucleotide sequence encoding animmunomodulatory polypeptide described herein. For example, apolynucleotide may be a polynucleotide comprising at least 70% sequenceidentity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% or higher, sequence identity compared to a reference polynucleotidesequence such as a sequence encoding an antibody described herein, usingthe methods described herein, (e.g., BLAST analysis using standardparameters, as described below). One skilled in this art will recognizethat these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like.

Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the binding affinity of the antibody encoded by the variantpolynucleotide is not substantially diminished relative to an antibodyencoded by a polynucleotide sequence specifically set forth herein.

In certain other related embodiments, polynucleotide fragments maycomprise or consist essentially of various lengths of contiguousstretches of sequence identical to or complementary to a sequenceencoding an immunomodulatory polypeptide as described herein. Forexample, polynucleotides are provided that comprise or consistessentially of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of a sequences the encodes an immunomodulatory polypeptide,or variant thereof, disclosed herein as well as all intermediate lengthsthere between. It will be readily understood that “intermediatelengths”, in this context, means any length between the quoted values,such as 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152,153, etc.; including all integers through 200-500; 500-1,000, and thelike. A polynucleotide sequence as described here may be extended at oneor both ends by additional nucleotides not found in the native sequence.This additional sequence may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides at either end of apolynucleotide encoding an immunomodulatory polypeptide described hereinor at both ends of a polynucleotide encoding an immunomodulatorypolypeptide described herein.

In another embodiment, polynucleotides are provided that are capable ofhybridizing under moderate to high stringency conditions to apolynucleotide sequence encoding an immunomodulatory polypeptide, orvariant thereof, provided herein, or a fragment thereof, or acomplementary sequence thereof. Hybridization techniques are well knownin the art of molecular biology. For purposes of illustration, suitablemoderately stringent conditions for testing the hybridization of apolynucleotide as provided herein with other polynucleotides includeprewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0);hybridizing at 50° C.-60° C., 5×SSC, overnight; followed by washingtwice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSCcontaining 0.1% SDS. One skilled in the art will understand that thestringency of hybridization can be readily manipulated, such as byaltering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60° C.-65° C. or 65° C.-70° C.

In certain embodiments, the polynucleotides described above, e.g.,polynucleotide variants, fragments and hybridizing sequences, encodeimmunomodulatory polypeptides that bind neutrophils. In otherembodiments, such polynucleotides encode immunomodulatory polypeptides,or variants thereof, that bind to neutrophils at least about 50%, atleast about 70%, and in certain embodiments, at least about 90% as wellas an immunomodulatory polypeptide sequence specifically set forthherein (e.g., PeptideX2). In further embodiments, such polynucleotidesencode immunomodulatory polypeptides, or variants thereof, that bind toneutrophils with greater affinity than the immunomodulatory polypeptidesset forth herein, for example, that bind quantitatively at least about105%, 106%, 107%, 108%, 109%, or 110% as well as an immunomodulatorypeptide sequence specifically set forth herein.

As described elsewhere herein, determination of the three-dimensionalstructures of representative polypeptides (e.g., PeptideX2, IgX oranother PeptideX2 sequence-containing polypeptide of 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more aminoacids) may be made through routine methodologies such that substitution,addition, deletion or insertion of one or more amino acids with selectednatural or non-natural amino acids can be virtually modeled for purposesof determining whether a so derived structural variant retains thespace-filling properties of presently disclosed species. A variety ofcomputer programs are known to the skilled artisan for determiningappropriate amino acid substitutions (or appropriate polynucleotidesencoding the amino acid sequence) within an antibody such that, forexample, affinity is maintained or better affinity is achieved.

The polynucleotides described herein, or fragments thereof, regardlessof the length of the coding sequence itself, may be combined with otherDNA sequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, illustrative polynucleotide segments with totallengths of about 10,000, about 5000, about 3000, about 2,000, about1,000, about 500, about 200, about 100, about 50 base pairs in length,and the like, (including all intermediate lengths) are contemplated tobe useful.

When comparing polynucleotide sequences, two sequences are said to be“identical” if the sequence of nucleotides in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990);Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., CABIOS 5:151-153 (1989); Myers, E. W.and Müller W., CABIOS 4:11-17 (1988); Robinson, E. D., Comb. Theor11:105 (1971); Santou, N. Nes, M., Mol. Biol. Evol. 4:406-425 (1987);Sneath, P. H. A. and Sokal, R. R., Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.(1973); Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad., Sci. USA80:726-730 (1983).

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman, Add.APL. Math 2:482 (1981), by the identity alignment algorithm of Needlemanand Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similaritymethods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444(1988), by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nucl.Acids Res. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol.215:403-410 (1990), respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity among two or more the polynucleotides. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

In certain embodiments, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) of 20 percent or less, usually 5 to 15percent, or 10 to 12 percent, as compared to the reference sequences(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The percentage is calculated by determining thenumber of positions at which the identical nucleic acid bases occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thereference sequence (i.e., the window size) and multiplying the resultsby 100 to yield the percentage of sequence identity.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode an immunomodulatory peptide as described herein,or an antibody that specifically binds to such a peptide, as describedherein. Some of these polynucleotides bear minimal sequence identity tothe nucleotide sequence of the native or original polynucleotidesequence that encode immunomodulatory polypeptides described herein.Nonetheless, polynucleotides that vary due to differences in codon usageare expressly contemplated by the present disclosure. In certainembodiments, sequences that have been codon-optimized for mammalianexpression are specifically contemplated.

Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, may be employed for thepreparation of variants and/or derivatives of the immunomodulatorypolypeptides described herein. By this approach, specific modificationsin a polypeptide sequence can be made through mutagenesis of theunderlying polynucleotides that encode them. These techniques provides astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments, the inventors contemplate the mutagenesis of thepolynucleotide sequences that encode an immunomodulatory polypeptidedisclosed herein, or a variant thereof, to alter one or more propertiesof the encoded polypeptide, such as the binding affinity of the peptideor the variant thereof, or the immunosuppressive or immunostimulatoryeffects. The techniques of site-specific mutagenesis are well-known inthe art, and are widely used to create variants of both polypeptides andpolynucleotides. For example, site-specific mutagenesis is often used toalter a specific portion of a DNA molecule. In such embodiments, aprimer comprising typically about 14 to about 25 nucleotides or so inlength is employed, with about 5 to about 10 residues on both sides ofthe junction of the sequence being altered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In another approach for the production of polypeptide variants,recursive sequence recombination, as described in U.S. Pat. No.5,837,458, may be employed. In this approach, iterative cycles ofrecombination and screening or selection are performed to “evolve”individual polynucleotide variants having, for example, increasedbinding affinity. Certain embodiments also provide constructs in theform of plasmids, vectors, transcription or expression cassettes whichcomprise at least one polynucleotide as described herein.

According to certain related embodiments there is provided a recombinanthost cell which comprises one or more constructs as described herein; anucleic acid encoding immunomodulatory polypeptide or variant thereof;and a method of producing of the encoded product, which method comprisesexpression from encoding nucleic acid therefor. Expression mayconveniently be achieved by culturing under appropriate conditionsrecombinant host cells containing the nucleic acid. Following productionby expression, an immunomodulatory polypeptide may be isolated and/orpurified using any suitable technique, and then used as desired.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common, preferredbacterial host is E. coli.

The expression of peptides in prokaryotic cells such as E. coli is wellestablished in the art. For a review, see for example Pluckthun, A.Bio/Technology 9: 545-551 (1991). Expression in eukaryotic cells inculture is also available to those skilled in the art as an option forproduction of immunomodulatory polypeptides, see recent reviews, forexample Ref, M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J.et al. (1995) Curr. Opinion Biotech 6: 553-560.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992,or subsequent updates thereto.

The term “host cell” is used to refer to a cell into which has beenintroduced, or which is capable of having introduced into it, a nucleicacid sequence encoding one or more of the herein describedimmunomodulatory polypeptides, and which further expresses or is capableof expressing a selected gene of interest, such as a gene encoding anyherein described immunomodulatory polypeptide. The term includes theprogeny of the parent cell, whether or not the progeny are identical inmorphology or in genetic make-up to the original parent, so long as theselected gene is present. Accordingly there is also contemplated amethod comprising introducing such nucleic acid into a host cell. Theintroduction may employ any available technique. For eukaryotic cells,suitable techniques may include calcium phosphate transfection,DEAE-Dextran, electroporation, liposome-mediated transfection andtransduction using retrovirus or other virus, e.g. vaccinia or, forinsect cells, baculovirus. For bacterial cells, suitable techniques mayinclude calcium chloride transformation, electroporation andtransfection using bacteriophage. The introduction may be followed bycausing or allowing expression from the nucleic acid, e.g. by culturinghost cells under conditions for expression of the gene. In oneembodiment, the nucleic acid is integrated into the genome (e.g.chromosome) of the host cell. Integration may be promoted by inclusionof sequences which promote recombination with the genome, inaccordance—with standard techniques.

The present invention also provides, in certain embodiments, a methodwhich comprises using a construct as stated above in an expressionsystem in order to express a particular polypeptide such as animmunomodulatory polypeptide as described herein. The term“transduction” is used to refer to the transfer of genes from onebacterium to another, usually by a phage. “Transduction” also refers tothe acquisition and transfer of eukaryotic cellular sequences byretroviruses. The term “transfection” is used to refer to the uptake offoreign or exogenous DNA by a cell, and a cell has been “transfected”when the exogenous DNA has been introduced inside the cell membrane. Anumber of transfection techniques are well known in the art and aredisclosed herein. See, e.g., Graham et al., 1973, Virology 52:456;Sambrook et al., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, ColdSpring Harbor Laboratories; Davis et al., 1986, BASIC METHODS INMOLECULAR BIOLOGY, Elsevier; and Chu et al., 1981, Gene 13:197. Suchtechniques can be used to introduce one or more exogenous DNA moietiesinto suitable host cells.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, or may be maintained transiently as an episomal element withoutbeing replicated, or may replicate independently as a plasmid. A cell isconsidered to have been stably transformed when the DNA is replicatedwith the division of the cell. The term “naturally occurring” or“native” when used in connection with biological materials such asnucleic acid molecules, polypeptides, host cells, and the like, refersto materials which are found in nature and are not manipulated by ahuman. Similarly, “non-naturally occurring” or “non-native” as usedherein refers to a material that is not found in nature or that has beenstructurally modified or synthesized by a human.

Certain embodiments contemplated herein include antisense-based nucleicacid technologies that may be implemented in a manner that specificallyalters (e.g., increases or decreases in a statistically significantmanner) expression of a PeptideX2-encoding polynucleotide, or of apolynucleotide that encodes a polypeptide as provided herein whichcomprises the PeptideX2 [SEQ ID NO:2]amino acid sequence, such as apolynucleotide that encodes any of the polypeptides having amino acidsequences set forth in SEQ ID NOS:2 and 4-105. Such antisense-basedtechnologies include RNA interference, ribozymes and antisense nucleicacids.

RNA interference (RNAi) is a polynucleotide sequence-specific,post-transcriptional gene silencing mechanism effected bydouble-stranded RNA that results in degradation of a specific messengerRNA (mRNA), thereby reducing the expression of a desired targetpolypeptide encoded by the mRNA (see, e.g., WO 99/32619; WO 01/75164;U.S. Pat. No. 6,506,559; Fire et al., Nature 391:806-11 (1998); Sharp,Genes Dev. 13:139-41 (1999); Elbashir et al. Nature 411:494-98 (2001);Harborth et al., J. Cell Sci. 114:4557-65 (2001)). RNAi is mediated bydouble-stranded polynucleotides as also described hereinbelow, forexample, double-stranded RNA (dsRNA), having sequences that correspondto exonic sequences encoding portions of the polypeptides for whichexpression is compromised. RNAi reportedly is not effected bydouble-stranded RNA polynucleotides that share sequence identity withintronic or promoter sequences (Elbashir et al., 2001). RNAi pathwayshave been best characterized in Drosophila and Caenorhabditis elegans,but “small interfering RNA” (siRNA) polynucleotides that interfere withexpression of specific polypeptides in higher eukaryotes such as mammals(including humans) have also been described (e.g., Tuschl, 2001Chembiochem. 2:239-245; Sharp, 2001 Genes Dev. 15:485; Bernstein et al.,2001 RNA 7:1509; Zamore, 2002 Science 296:1265; Plasterk, 2002 Science296:1263; Zamore 2001 Nat. Struct. Biol. 8:746; Matzke et al., 2001Science 293:1080; Scadden et al., 2001 EMBO Rep. 2:1107) andsubsequently elaborated upon.

According to a current non-limiting model, the RNAi pathway is initiatedby ATP-dependent, processive cleavage of long dsRNA into double-strandedfragments of about 18-27 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, etc.)nucleotide base pairs in length, called small interfering RNAs (siRNAs)(see review by Hutvagner et al., Curr. Opin. Gen. Dev. 12:225-32 (2002);Elbashir et al., 2001; Nykänen et al., Cell 107:309-21 (2001); Zamore etal., Cell 101:25-33 (2000); Bass, Cell 101:235-38 (2000)). InDrosophila, an enzyme known as “Dicer” cleaves the longerdouble-stranded RNA into siRNAs; Dicer belongs to the RNase III familyof dsRNA-specific endonucleases (WO 01/68836; Bernstein et al., Nature409:363-66 (2001)). Further according to this non-limiting model, thesiRNA duplexes are incorporated into a protein complex, followed byATP-dependent unwinding of the siRNA, which then generates an activeRNA-induced silencing complex (RISC) (WO 01/68836). The complexrecognizes and cleaves a target RNA that is complementary to the guidestrand of the siRNA, thus interfering with expression of a specificprotein (Hutvagner et al., supra).

In C. elegans and Drosophila, RNAi may be mediated by longdouble-stranded RNA polynucleotides (WO 99/32619; WO 01/75164; Fire etal., 1998; Clemens et al., Proc. Natl. Acad. Sci. USA 97:6499-6503(2000); Kisielow et al., Biochem. J. 363:1-5 (2002); see also WO01/92513 (RNAi-mediated silencing in yeast)). In mammalian cells,however, transfection with long dsRNA polynucleotides (i.e., greaterthan 30 base pairs) leads to activation of a non-specific sequenceresponse that globally blocks the initiation of protein synthesis andcauses mRNA degradation (Bass, Nature 411:428-29 (2001)). Transfectionof human and other mammalian cells with double-stranded RNAs of about18-27 nucleotide base pairs in length interferes in a sequence-specificmanner with expression of particular polypeptides encoded by messengerRNAs (mRNA) containing corresponding nucleotide sequences (WO 01/75164;Elbashir et al., 2001; Elbashir et al., Genes Dev. 15:188-200 (2001));Harborth et al., J. Cell Sci. 114:4557-65 (2001); Carthew et al., Curr.Opin. Cell Biol. 13:244-48 (2001); Mailand et al., Nature Cell Biol.Advance Online Publication (Mar. 18, 2002); Mailand et al. 2002 NatureCell Biol. 4:317).

siRNA polynucleotides may offer certain advantages over otherpolynucleotides known to the art for use in sequence-specific alterationor modulation of gene expression to yield altered levels of an encodedpolypeptide product. These advantages include lower effective siRNApolynucleotide concentrations, enhanced siRNA polynucleotide stability,and shorter siRNA polynucleotide oligonucleotide lengths relative tosuch other polynucleotides (e.g., antisense, ribozyme or triplexpolynucleotides).

By way of a brief background, “antisense” polynucleotides bind in asequence-specific manner to target nucleic acids, such as mRNA or DNA,to prevent transcription of DNA or translation of the mRNA (see, e.g.,U.S. Pat. Nos. 5,168,053; 5,190,931; 5,135,917; 5,087,617; see also,e.g., Clusel et al., 1993 Nucl. Acids Res. 21:3405-11, describing“dumbbell” antisense oligonucleotides). “Ribozyme” polynucleotides canbe targeted to any RNA transcript and are capable of catalyticallycleaving such transcripts, thus impairing translation of mRNA (see,e.g., U.S. Pat. Nos. 5,272,262; 5,144,019; and 5,168,053, 5,180,818,5,116,742 and 5,093,246; U.S. 2002/193579). “Triplex” DNA moleculesrefers to single DNA strands that bind duplex DNA to form a colineartriplex molecule, thereby preventing transcription (see, e.g., U.S. Pat.No. 5,176,996, describing methods for making synthetic oligonucleotidesthat bind to target sites on duplex DNA). Such triple-strandedstructures are unstable and form only transiently under physiologicalconditions.

Because single-stranded polynucleotides do not readily diffuse intocells and are therefore susceptible to nuclease digestion, developmentof single-stranded DNA for antisense or triplex technologies oftenrequires chemically modified nucleotides to improve stability andabsorption by cells. siRNAs, by contrast, are readily taken up by intactcells, are effective at interfering with the expression of specificpolypeptides at concentrations that are several orders of magnitudelower than those required for either antisense or ribozymepolynucleotides, and do not require the use of chemically modifiednucleotides.

It will be appreciated that the practice of the several embodiments ofthe present invention will employ, unless indicated specifically to thecontrary, conventional methods in virology, immunology, microbiology,molecular biology and recombinant DNA techniques that are within theskill of the art, and many of which are described below for the purposeof illustration. Such techniques are explained fully in the literature.See, e.g., Current Protocols in Molecular Biology or Current Protocolsin Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al.,Short Protocols in Molecular Biology, 3^(rd) ed., Wiley & Sons, 1995;Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rdEdition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual(1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984) and other like references.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. These and relatedtechniques and procedures may be generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of, molecular biology, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for recombinant technology,molecular biological, microbiological, chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Throughout this specification, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element or integer or group of elements or integers but notthe exclusion of any other element or integer or group of elements orintegers. Each embodiment in this specification is to be applied mutatismutandis to every other embodiment unless expressly stated otherwise.

Compositions and Methods of Use

The present disclosure provides compositions comprising the hereindescribed immunomodulatory polypeptides and variants thereof, which inpreferred embodiments may comprise a PeptideX2-containing polypeptide asprovided herein (e.g., a polypeptide comprising SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:105 or SEQ ID NO:106) and/or an antibody as providedherein that specifically binds to such a PeptideX2-containingpolypeptide, and also provides administration of such compositions in avariety of therapeutic settings.

Administration of the immunomodulatory polypeptides, or antibodiesspecific therefor, described herein, in pure form or in an appropriatepharmaceutical composition, can be carried out via any of the acceptedmodes of administration of agents for serving similar utilities. Thepharmaceutical compositions can be prepared by combining animmunomodulatory polypeptide or immunomodulatory polypeptide-containingcomposition or an antibody specific for PeptideX2 with an appropriatephysiologically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid ormicroparticle—(e.g., microdroplet) containing gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, and aerosols.In addition, other pharmaceutically active ingredients (including otherimmunosuppressive agents as described elsewhere herein) and/or suitableexcipients such as salts, buffers and stabilizers may, but need not, bepresent within the composition. Administration may be achieved by avariety of different routes, including oral, parenteral, nasal,intravenous, intradermal, subcutaneous or topical.

Preferred modes of administration depend upon the nature of thecondition to be treated or prevented, which in certain embodiments willrefer to a deleterious or clinically undesirable condition the extent,severity, likelihood of occurrence and/or duration of which may bedecreased (e.g., reduced in a statistically significant manner relativeto an appropriate control situation such as an untreated control)according to certain methods provided herein. An amount that, followingadministration, detectably reduces, inhibits, prevents, decreases theseverity or likelihood of occurrence of, or delays such a condition, forinstance, the onset or exacerbation of sepsis, or the rejection of atransplant such as an organ allograft or bone marrow transplant, or thepartial or complete reduction of a tumor burden, is consideredeffective. Persons skilled in the relevant arts will be familiar withany number of diagnostic, surgical and/or other clinical criteria thatmay indicate the clinical appropriateness of, and/or to which can beadapted, administration of the immunomodulatory compositions describedherein. See, e.g., Faix, 2013 Crit. Rev. Clin. Lab. Sci. 50(1):23-36(“Biomarkers of Sepsis”); Wiersinga et al., 2014 Virulence 5(1):36-44(“Host innate immune responses to sepsis”); Hotchkiss et al., 2013 Nat.Rev. Immunol. 13:862; Aziz et al., 2013 J. Leukoc. Biol. 93(3):329;Beyrau et al., 2012 Open Biol. 2:120134; Fry, 2012 Amer. Surg. 78:1;Kellum et al., 2007 Arch. Intern. Med. 167(15):1655; Remick, 2007 Am. J.Pathol. 170(5):1435; Hotchkiss et al., 2003 New Engl. J. Med.348:138-150; Humar et al., Atlas of Organ Transplantation, 2006,Springer; Kuo et al., Comprehensive Atlas of Transplantation, 2004Lippincott, Williams & Wilkins; Gruessner et al., Living Donor OrganTransplantation, 2007 McGraw-Hill Professional; Antin et al., Manual ofStem Cell and Bone Marrow Transplantation, 2009 Cambridge UniversityPress; Wingard et al. (Ed.), Hematopoietic Stem Cell Transplantation: AHandbook for Clinicians, 2009 American Association of Blood Banks.

Neutrophil roles in sepsis have been described and include an earlydominant “hyperinflammatory phase” of potent TLR-mediated induction ofinflammatory cytokine release (e.g., IL-6, TNFα, IL-1) following PAMPrecognition, which subsequently gives way to a concurrent butlater-dominant “hypoinflammatory phase” of TLR-mediatedimmunosuppression following DAMP recognition, this latter phasecharacterized by apoptotic depletion of myeloid as well as lymphoidadaptive immune cells, release by neutrophils of the immunosuppressivecytokine IL-10, and reduced levels of inflammatory cytokines (e.g.,Faix, 2013 Crit. Rev. Clin. Lab. Sci. 50(1):23-36; Wiersinga et al.,2014 Virulence 5(1):36-44; Fournier, 2013 Front. Cell. Infect.Microbiol. 2: Art. 167; Hotchkiss et al., 2013 Nat. Rev. Immunol.13:862; Aziz et al., 2013 J. Leukoc. Biol. 93(3):329; Beyrau et al.,2012 Open Biol. 2:120134; Fry, 2012 Amer. Surg. 78:1; Kellum et al.,2007 Arch. Intern. Med. 167(15):1655; Remick, 2007 Am. J. Pathol.170(5): 1435; Hotchkiss et al., 2003 New Engl. J. Med. 348:138-150; Menget al., 2004 J. Clin. Invest. 113(10):1473-1481; Decker, 2004 J. Clin.Invest. 113:1387-1389); Navarini et al., 2009 Proc. Nat. Acad. Sci. USA106:7107-7112; Roger et al., 2009 Proc. Nat. Acad. Sci. USA 106:6889;Alves-Filho et al., 2009 Proc. Nat. Acad. Sci. USA 106:4018; Zou et al.,2011 Shock 36:370; Castoldi et al, 2012 PLoS ONE 7(5):e37584; Pene etal., 2009 Infect. Immun. 77(12):5651).

As described herein for the first time and presented in greater detailbelow, the presently provided peptide X2 directly bound to asubpopulation of neutrophils and also, after being contacted withneutrophil-containing peripheral blood leukocyte preparations, induced,inter alia, elaboration of IL-6 and IL-10. PeptideX2 is also shown herefor the first time to be capable of delivering a specifictranscriptional activation signal to innate immune system cells, viabinding interactions with human or murine TLR2 and/or TLR4 (but notother human or murine TLRs) at discrete and detectable signaling levelsthat were nevertheless well below the levels delivered by natural PAMPligands for these TLRs. PeptideX2 induced peripheral blood white cellsto release the pro-inflammatory cytokine TNFα without compromising theability of these immune system cells to inhibit bacterial (S. aureus)growth in an in vitro assay.

By these effects on local and/or systemic immunologic status (e.g.,hyperinflammatory vs. hypoinflammatory, altered cytokine profile, etc.),which may vary as a function of PeptideX2 dosage parameters (e.g.,concentration, timing, absence or presence of competing TLR2/4 ligandssuch as PAMPs or DAMPs, activation status of target cells, host clinicalstatus, valency, etc.), the herein described PeptideX2 and IgXpolypeptides and/or antibodies specific for such PeptideX2 or IgXpolypeptides are thus believed according to non-limiting theory toprovide useful immunomodulatory properties. The presently disclosedPeptideX2 and IgX polypeptides afford such properties through theiralteration (e.g., statistically significant increases or decreases) inthe activity levels of one or more cellular regulators of immune status,which according to certain preferred embodiments and further accordingto non-limiting theory relate to unexpected advantages that are obtainedby their dual functioning as (i) weak agonists of TLR2 and/or TLR4,through which biological signals are transduced that are qualitativelyand quantitatively less profound than TLR2/TLR4 activation signals thatare transduced in response to PAMPs or DAMPS, and (ii) antagonists ofPAMPs and/or DAMPs by virtue of their competitive, albeit low affinity,binding to TLR2 and TLR4. In this respect, the presently disclosedPeptideX2 and IgX immunomodulatory polypeptides surprisingly permit theinnate immune system to mediate a moderate inflammatory response insteadof the exuberant hyperinflammatory reaction that characterizes sepsis,without driving the immune system to the immunosuppressed state thatotherwise often subsequently predominates later stages of sepsis.

Preliminary animal and human trials may be performed to test the safetyand efficacy of PeptideX2 for the treatment of sepsis. For example andby way of non-limiting illustration, in a proposed human trial ofPeptideX2 in sepsis, the immunomodulatory peptide is administeredintravenously to achieve a serum concentration of about 50-100 ug/ml.Assuming a total body distribution volume of 6 liters, administration toa patient of 500 mg of PeptideX2 over 1 hour would represent anestimated plasma concentration of 80 ug/ml. As a reference forcomparison, in the treatment of rheumatoid arthritis, the TNFαantagonist infliximab (Remicade®, Janssen Biotech Inc.) is typicallyadministered intravenously to a patient weighing 70 kg at 3-10mg/kg/dose, which would be calculated as delivery of 300 mg to 700 mg ofinfliximab as an infusion.

In another embodiment, the amount administered is sufficient to increasethe rate of embryo implantation. In certain embodiments, theimmunomodulatory polypeptide is administered to in vitro fertilization(IVF) patients (which may include a pseudopregnant patient such as asurrogate mother), and/or contacted with an IVF-generated embryo invitro, to increase, promote or permit implantation of the embryo. Inother embodiments, the immunomodulatory polypeptide is administered toan individual trying to get pregnant with or without prior diagnosedfertility difficulties. The effectiveness of an immunomodulatorypolpeptide to modulate the immune response, and thereby modulate embryoimplantation, can be determined using assays known in the art, such as,for example, the “PIF assays” described in U.S. Pat. Nos. 5,646,003 and5,981,198 and PCT Application Publication Nos. WO 2003/004601 and WO2005/040196, the disclosures of which are incorporated herein byreference in their entirety. Principles and practices in reproductivemedicine are known to skilled clinicians, who will appreciate thefactors involved in adaptation of the present disclosure to the clinicalsetting. See, e.g., Lebovic et al., Reproductive Endocrinology andInfertility: Handbook for Clinicians, 2005 Scrub Hill Press; Botros etal., Infertility and Assisted Reproduction, 2008 Cambridge Univ. Press;Greene et al., Creasy and Resnick's Maternal-Fetal Medicine: Principleand Practice, 2008 Saunders Publishing; Cunningham et al., Williams'Obstetrics-23rd Ed. 2009 McGraw-Hill Professional.

In other embodiments, the amount administered is sufficient to result inclinically relevant reduction in symptoms of preeclampsia,hemolysis-elevated liver enzymes-low platelet count (HELLP) syndrome andeclampsia, such as, but not limited to, reduction of any one or more ofhypertension, edema of the hands and/or face, proteinuria, sudden weightgain, nausea, vomiting, abdominal pain, shoulder pain, lower back pain,muscle aches or pains, headache, changes in vision, blurry vision,hyperreflexia, racing pulse, mental confusion, anxiety, shortness ofbreath, hemolysis, elevated liver enzymes, low platelet count, decreasedurine output, fatigue, fluid retention, nosebleeds, enlarged liver andseizures or convulsions.

In other embodiments, the amount of PeptideX2-containing polypeptide(e.g., a polypeptide comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:105or SEQ ID NO:106) or specific anti-PeptideX2 antibody that isadministered is sufficient to result in clinically relevant reduction insymptoms of autoimmune diseases, including but not limited to rheumatoidarthritis (RA), systemic lupus erythematosus (SLE), inflammatory boweldisease (IBD), psoriatic arthritis, Crohn's disease, ulcerative colitis,seronegative spondyloarthropathies, Behcet's disease, vasculitis, andother autoimmune diseases. Reduction in RA symptoms may be evidenced,for example by way of illustration and not limitation, as reduction ofany one or more of fatigue, loss of appetite, low fever, swollen glands,weakness, swollen joints, joint pain, morning stiffness, warm, tender,and stiff joints when not used for as little as an hour, bilateral jointpain (fingers (but not the fingertips), wrists, elbows, shoulders, hips,knees, ankles, toes, jaw, and neck may be affected); loss of range ofmotion of affected joints, pleurisy, eye burning, eye itching, eyedischarge, nodules under the skin, numbness, tingling, or burning in thehands and feet. Criteria for diagnosis and clinical monitoring of RApatients are well known to those skilled in the relevant art. See, e.g.,Hochberg et al., Rheumatology, 2010 Mosby; Firestein et al., Textbook ofRheumatology, 2008 Saunders. Criteria for diagnosis and clinicalmonitoring of patients having RA and/or other autoimmune diseases arealso well known to those skilled in the relevant art. See, e.g., Petrov,Autoimmune Disorders: Symptoms, Diagnosis and Treatment, 2011 NovaBiomedical Books; Mackay et al. (Eds.), The Autoimmune Diseases-FourthEdition, 2006 Academic Press; Brenner (Ed.), Autoimmune Diseases:Symptoms, Diagnosis and Treatment, 2011 Nova Science Pub. Inc.

Certain embodiments contemplate method of treating a malignantcondition, comprising administering to a subject having or suspected ofhaving a malignancy a composition that comprises a therapeuticallyeffective amount of an immunomodulatory polypeptide that compriseseither the amino acid sequence set forth in SEQ ID NO:2 or the aminoacid sequence set forth in SEQ ID NO:106, or the use of antibodies thatspecifically bind to peptideX2, IgX, or other peptideX2-containingpolypeptides (e.g., a polypeptide comprising SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:105 or SEQ ID NO:106) and thereby treating the malignantcondition. According to certain such embodiments, the peptide thatcomprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:105 or SEQ ID NO:106, orthe antibody that is capable of specifically binding to a peptide thatcomprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:105 or SEQ ID NO:106,promotes altered (e.g., increased or decreased in a statisticallysignificant manner) immunological activity that results in immunesystem-potentiated killing of tumor cells and/or inhibition of tumormetastasis. By way of non-limiting theory, such altered immunologicalactivity may be induced or promoted by binding of the herein describedimmunomodulatory polypeptide (e.g., the polypeptide which comprises SEQID NO:2, SEQ ID NO:4, SEQ ID NO:105 or SEQ ID NO:106) to one or morecognate receptors on immunologically active cells including neutrophils,or by interference with such binding by antibody blockade when ananti-PeptideX2 antibody is administered.

Neutrophil roles in cancer cell rejection and metastasis have beendescribed, including in breast cancer, ovarian cancer, adenoma,colorectal, gastric, lung, prostate and hepatocellular carcinoma,melanoma, and hematologic (e.g., leukemia, lymphoma) and othermalignancies (e.g., DiCarlo et al., 2001 Blood 97; 339; Mantovani etal., 2011 Nat. Rev. Immunol. 11:519; Gregory et al., 2011 Canc. Res.71:2411; De Larco et al., 2004 Clin. Canc. Rec. 10:4895), as haveanti-tumor effects of the known neutrophil products IL-6 and IL-10(e.g., Li et al., 2010 Canc. Chemother. Pharmacol. 66:981; Mumm et al.,2011 Canc. Cell 20:781). As described herein for the first time andpresented in greater detail below, the presently provided peptide X2directly binds to neutrophils and also, after being contacted withneutrophil-containing peripheral blood leukocyte preparations, induces,inter alia, elaboration of IL-6 and IL-10. This effect on a local and/orsystemic cytokine profile is thus believed to provide immunomodulatoryproperties of the herein described PeptideX2 and IgX polypeptides and/orof antibodies specific for such PeptideX2 or IgX polypeptides, throughtheir alteration (e.g., statistically significant increases ordecreases) in the activity levels of one or more cellular regulators ofimmune status. (See, e.g., DiCarlo et al., 2001 Blood 97; 339; Mantovaniet al., 2011 Nat. Rev. Immunol. 11:519.)

The presence of a malignant condition in a subject refers to thepresence of dysplastic, cancerous and/or transformed cells in thesubject, including, for example neoplastic, tumor, non-contact inhibitedor oncogenically transformed cells, or the like (e.g., carcinomas suchas adenocarcinoma, squamous cell carcinoma, small cell carcinoma, oatcell carcinoma, etc., sarcomas such as chondrosarcoma, osteosarcoma,etc.) which are known to the art and for which criteria for diagnosisand classification are established (e.g., Hanahan and Weinberg, 2011Cell 144:646; Hanahan and Weinberg 2000 Cell 100:57; Cavallo et al.,2011 Canc. Immunol. Immunother. 60:319; Kyrigideis et al., 2010 J.Carcinog. 9:3) In preferred embodiments contemplated by the presentinvention, for example, such cancer cells may be cells of mixed lineageleukemia, esophageal cancer, ovarian cancer, prostate cancer, kidneycancer, colon cancer, liver cancer, stomach cancer, breast cancer andpancreatic cancer, and other solid cancers. The precise dosage andduration of treatment is a function of the condition or disease beingtreated and may be determined empirically using known testing protocolsor by testing the compositions in model systems known in the art andextrapolating therefrom. Controlled clinical trials may also beperformed. Dosages may also vary with the severity of the condition tobe alleviated. A pharmaceutical composition is generally formulated andadministered to exert a therapeutically useful effect while minimizingundesirable side effects. The composition may be administered one time,or may be divided into a number of smaller doses to be administered atintervals of time. For any particular subject, specific dosage regimensmay be adjusted over time according to the individual need.

The immunomodulatory-containing compositions may be administered aloneor in combination with other known immunosuppressive treatments, such asmonoclonal antibodies to lymphocytes and cytokine receptors (e.g.,anti-IL-2Rα), calcineurin inhibitors (e.g., cyclosporine andtacrolimus), and cytokine receptor signal transduction inhibitors (e.g.,sirolimus). The compositions may also be administered in combinationwith antibiotics.

Typical routes of administering these and related pharmaceuticalcompositions thus include, without limitation, oral, topical,transdermal, inhalation, parenteral, sublingual, buccal, rectal,vaginal, and intranasal. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques. Pharmaceutical compositions accordingto certain embodiments of the present invention are formulated so as toallow the active ingredients contained therein to be bioavailable uponadministration of the composition to a patient. Compositions that willbe administered to a subject or patient may take the form of one or moredosage units, where for example, a tablet may be a single dosage unit,and a container of a herein described immunomodulatory polypeptide inaerosol form may hold a plurality of dosage units. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington: The Science andPractice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy andScience, 2000). The composition to be administered will, in any event,contain a therapeutically effective amount of an immunomodulatorypolypeptide of the present disclosure, for treatment of a disease orcondition of interest in accordance with teachings herein.

A pharmaceutical composition may be in the form of a solid or liquid. Inone embodiment, the carrier(s) are particulate, so that the compositionsare, for example, in tablet or powder form. The carrier(s) may beliquid, with the compositions being, for example, an oral oil,injectable liquid or an aerosol, which is useful in, for example,inhalatory administration. When intended for oral administration, thepharmaceutical composition is preferably in either solid or liquid form,where semi-solid, semi-liquid, suspension and gel forms are includedwithin the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like. Such a solid compositionwill typically contain one or more inert diluents or edible carriers. Inaddition, one or more of the following may be present: binders such ascarboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gumtragacanth or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin; a flavoring agent such as peppermint,methyl salicylate or orange flavoring; and a coloring agent. When thepharmaceutical composition is in the form of a capsule, for example, agelatin capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions, whether they be solutions,suspensions or other like form, may include one or more of the followingadjuvants: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or diglycerides whichmay serve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. Physiological saline isa preferred adjuvant. An injectable pharmaceutical composition ispreferably sterile.

A liquid pharmaceutical composition intended for either parenteral ororal administration should contain an amount of an immunomodulatorypolypeptide as herein disclosed such that a suitable dosage will beobtained. Typically, this amount is at least 0.01% of theimmunomodulatory polypeptide in the composition. When intended for oraladministration, this amount may be varied to be between 0.1 and about70% of the weight of the composition. Certain oral pharmaceuticalcompositions contain between about 4% and about 75% of theimmunomodulatory polypeptide. In certain embodiments, pharmaceuticalcompositions and preparations according to the present invention areprepared so that a parenteral dosage unit contains between 0.01 to 10%by weight of the immunomodulatory polypeptide prior to dilution.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. The pharmaceutical composition may beintended for rectal administration, in the form, for example, of asuppository, which will melt in the rectum and release the drug. Thecomposition for rectal administration may contain an oleaginous base asa suitable nonirritating excipient. Such bases include, withoutlimitation, lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition may include various materials, whichmodify the physical form of a solid or liquid dosage unit. For example,the composition may include materials that form a coating shell aroundthe active ingredients. The materials that form the coating shell aretypically inert, and may be selected from, for example, sugar, shellac,and other enteric coating agents. Alternatively, the active ingredientsmay be encased in a gelatin capsule. The pharmaceutical composition insolid or liquid form may include an agent that binds to theimmunomodulatory polypeptide of the invention and thereby assists in thedelivery of the compound. Suitable agents that may act in this capacityinclude monoclonal or polyclonal antibodies, one or more proteins or aliposome. The pharmaceutical composition may consist essentially ofdosage units that can be administered as an aerosol. The term aerosol isused to denote a variety of systems ranging from those of colloidalnature to systems consisting of pressurized packages. Delivery may be bya liquefied or compressed gas or by a suitable pump system thatdispenses the active ingredients. Aerosols may be delivered in singlephase, bi-phasic, or tri-phasic systems in order to deliver the activeingredient(s). Delivery of the aerosol includes the necessary container,activators, valves, subcontainers, and the like, which together may forma kit. One of ordinary skill in the art, without undue experimentationmay determine preferred aerosols.

The pharmaceutical compositions may be prepared by methodology wellknown in the pharmaceutical art. For example, a pharmaceuticalcomposition intended to be administered by injection can be prepared bycombining a composition that comprises an immunomodulatory polypeptideas described herein and optionally, one or more of salts, buffers and/orstabilizers, with sterile, distilled water so as to form a solution. Asurfactant may be added to facilitate the formation of a homogeneoussolution or suspension. Surfactants are compounds that non-covalentlyinteract with the peptide composition so as to facilitate dissolution orhomogeneous suspension of the immunomodulatory polypeptide in theaqueous delivery system.

The compositions are administered in a therapeutically effective amount,which will vary depending upon a variety of factors including theactivity of the specific compound (e.g., IgX or PeptideX2) employed; themetabolic stability and length of action of the compound; the age, bodyweight, general health, sex, and diet of the patient; the mode and timeof administration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy. Generally, a therapeutically effective daily dose is(for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (fora 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg(i.e., 3.5 g); more preferably a therapeutically effective dose is (fora 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg(i.e., 1.75 g).

Compositions comprising the immunomodulatory polypeptides of the presentdisclosure may also be administered simultaneously with, prior to, orafter administration of one or more other therapeutic agents. Suchcombination therapy may include administration of a singlepharmaceutical dosage formulation which contains a compound of theinvention and one or more additional active agents, as well asadministration of compositions comprising antibodies of the inventionand each active agent in its own separate pharmaceutical dosageformulation. For example, an immunomodulatory polypeptide as describedherein and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Similarly, an immunomodulatory polypeptide as describedherein and the other active agent can be administered to the patienttogether in a single parenteral dosage composition such as in a salinesolution or other physiologically acceptable solution, or each agentadministered in separate parenteral dosage formulations. Where separatedosage formulations are used, the compositions comprising antibodies andone or more additional active agents can be administered at essentiallythe same time, i.e., concurrently, or at separately staggered times,i.e., sequentially and in any order; combination therapy is understoodto include all these regimens.

Thus, in certain embodiments, also contemplated is the administration ofimmunomodulatory polypeptide compositions of this disclosure incombination with one or more other therapeutic agents. Such therapeuticagents may be accepted in the art as a standard treatment for aparticular disease state as described herein, such as rheumatoidarthritis, inflammation or preeclampsia. Exemplary therapeutic agentscontemplated include cytokines, growth factors, steroids, NSAIDs,DMARDs, anti-inflammatories, chemotherapeutics, or other active andancillary agents.

In various embodiments, the immunomodulatory polypeptides describedherein are conjugated to a detectable label that may be detecteddirectly or indirectly. In this regard, an immunomodulatory polypeptide“conjugate” refers to an immunomodulatory polypeptide that is covalentlylinked to a detectable label. In the present invention, DNA probes, RNAprobes, monoclonal antibodies, antigen-binding fragments thereof, andantibody derivatives thereof, such as a single-chain-variable-fragmentantibody or an epitope tagged antibody, may all be covalently linked toa detectable label. In “direct detection”, only one detectable antibodyis used, i.e., a primary detectable antibody. Thus, direct detectionmeans that the antibody that is conjugated to a detectable label may bedetected, per se, without the need for the addition of a second antibody(secondary antibody).

A “detectable label” is a molecule or material that can produce adetectable (such as visually, electronically or otherwise) signal thatindicates the presence and/or concentration of the label in a sample.When conjugated to a peptide, the detectable label can be used to locateand/or quantify the target to which the specific peptide is bound.Thereby, the presence and/or concentration of the target in a sample canbe detected by detecting the signal produced by the detectable label. Adetectable label can be detected directly or indirectly, and severaldifferent detectable labels conjugated to different specific-antibodiescan be used in combination to detect one or more targets.

Examples of detectable labels, which may be detected directly, includefluorescent dyes and radioactive substances and metal particles. Incontrast, indirect detection requires the application of one or moreadditional antibodies, i.e., secondary antibodies, after application ofthe primary antibody. Thus, the detection is performed by the detectionof the binding of the secondary antibody or binding agent to the primarydetectable antibody. Examples of primary detectable binding agents orantibodies requiring addition of a secondary binding agent or antibodyinclude enzymatic detectable binding agents and hapten detectablebinding agents or antibodies.

In some embodiments, the detectable label is conjugated to a nucleicacid polymer which comprises the first binding agent (e.g., in an ISH,WISH, or FISH process). In other embodiments, the detectable label isconjugated to an antibody which comprises the first binding agent (e.g.,in an IHC process).

Examples of detectable labels which may be conjugated toimmunomodulatory polypeptides used in the methods of the presentdisclosure include fluorescent labels, enzyme labels, radioisotopes,chemiluminescent labels, electrochemiluminescent labels, bioluminescentlabels, polymers, polymer particles, metal particles, haptens, and dyes.

Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5-or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoicacid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, anddyes such as Cy2, Cy3, and Cy5, optionally substituted coumarinincluding AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE)and allophycoerythrin (APC), Texas Red, Princeton Red, green fluorescentprotein (GFP) and analogues thereof, and conjugates of R-phycoerythrinor allophycoerythrin, inorganic fluorescent labels such as particlesbased on semiconductor material like coated CdSe nanocrystallites.

Examples of polymer particle labels include micro particles or latexparticles of polystyrene, PMMA or silica, which can be embedded withfluorescent dyes, or polymer micelles or capsules which contain dyes,enzymes or substrates.

Examples of metal particle labels include gold particles and coated goldparticles, which can be converted by silver stains. Examples of haptensinclude DNP, fluorescein isothiocyanate (FITC), biotin, and digoxigenin.Examples of enzymatic labels include horseradish peroxidase (HRP),alkaline phosphatase (ALP or AP), β-galactosidase (GAL),glucose-6-phosphate dehydrogenase, β-N-acetylglucosamimidase,β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase andglucose oxidase (GO). Examples of commonly used substrates forhorseradishperoxidase include 3,3′-diaminobenzidine (DAB),diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole(AEC), Benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR),Indophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphtol(CN), .alpha.-naphtol pyronin (.alpha.-NP), o-dianisidine (OD),5-bromo-4-chloro-3-indolylphosp-hate (BCIP), Nitro blue tetrazolium(NBT), 2-(p-iodophenyl)-3-p-nitropheny-I-5-phenyl tetrazolium chloride(INT), tetranitro blue tetrazolium (TNBT),5-bromo-4-chloro-3-indoxyl-beta-D-galactoside/ferro-ferricyanide(BCIG/FF).

Examples of commonly used substrates for Alkaline Phosphatase includeNaphthol-AS-B 1-phosphate/fast red TR (NABP/FR),Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR),Naphthol-AS-B1-phosphate/-fast red TR (NABP/FR),Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR),Naphthol-AS-B1-phosphate/new fuschin (NABP/NF), bromochloroindolylphosphate/nitroblue tetrazolium (BCIP/NBT),5-Bromo-4-chloro-3-indolyl-b-d-galactopyranoside (BCIG).

Examples of luminescent labels include luminol, isoluminol, acridiniumesters, 1,2-dioxetanes and pyridopyridazines. Examples ofelectrochemiluminescent labels include ruthenium derivatives. Examplesof radioactive labels include radioactive isotopes of iodide, cobalt,selenium, tritium, carbon, sulfur and phosphorous.

Detectable labels may be linked to the immunomodulatory polypeptidesdescribed herein or to any other molecule that specifically binds to abiological marker of interest, e.g., an antibody, a nucleic acid probe,or a polymer. Furthermore, one of ordinary skill in the art wouldappreciate that detectable labels can also be conjugated to second,and/or third, and/or fourth, and/or fifth binding agents or antibodies,etc. Moreover, the skilled artisan would appreciate that each additionalbinding agent or antibody used to characterize a biological marker ofinterest may serve as a signal amplification step. The biological markermay be detected visually using, e.g., light microscopy, fluorescentmicroscopy, electron microscopy where the detectable substance is forexample a dye, a colloidal gold particle, a luminescent reagent.Visually detectable substances bound to a biological marker may also bedetected using a spectrophotometer. Where the detectable substance is aradioactive isotope detection can be visually by autoradiography, ornon-visually using a scintillation counter. See, e.g., Larsson, 1988,Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, Fla.);Methods in Molecular Biology, vol. 80 1998, John D. Pound (ed.) (HumanaPress, Totowa, N.J.).

The invention further and in certain embodiments provides kits fordetecting immunomodulatory polypeptides (e.g., IgX or PeptideX2) orcells (e.g., neutrophils) in a sample, wherein the kits contain at leastone antibody, polypeptide, polynucleotide, vector or host cell asdescribed herein. In certain embodiments, a kit may comprise buffers,enzymes, labels, substrates, beads or other surfaces to which theantibodies of the invention are attached, and the like, and instructionsfor use.

EXAMPLES Example 1 Identification of IgX

The modulation of the immune response during pregnancy has been thefocus of numerous studies; however, the underlying mechanisms thatmodulate the maternal immune system to allow for implantation, placentalentrenchment, the balance between pro-inflammatory and anti-inflammatorysignals, and the eventual achievement of a full term pregnancy remainunclear. For example, it is unclear why a majority of women withrheumatoid arthritis experience disease amelioration during pregnancy.In order to identify naturally occurring immunoregulatory agents,placentas from healthy women were analyzed.

Placental Preparation

Placentas were obtained from healthy women using appropriate informedconsent. The umbilical cord and embryonic sac were removed, the placentawas washed generously in physiologic saline three times, and it washomogenized using a blender at medium speed for 30 seconds followed byone minute at slow speed. Samples were then placed into conical tubesand centrifuged at 1400 g for 10 minutes. Supernatants were removed andstored. The pelleted tissue component was prepared by adding 10 ml ofnanopure water to each individual conical tube, placing each tube onice, sonicating each conical tube with placental tissue for 30 seconds,allowing it to cool for 3 minutes, and then repeating three times. Next,50 cc of nanopure water was added to each conical tube and mixed tocreate a hypotonic solution and cause cell lysis. Each sample wasdiluted to 1:10,000 in merthiolate, incubated at 4° C. for 48 hours,then centrifuged at 1400 g for 25 minutes, and then the lysate wasaspirated from the conical tube and stored as −20° C.

IqX Preparation

Frozen lysate from the placental tissue was thawed and centrifuged10,000 g for 13 minutes. The resulting supernatant was collected, and500 mg EDTA (all reagents were from Sigma, St. Louis, Mo., unlessotherwise noted) was added to each tube and mixed well. The lysates werethen incubated at room temperature for 30 minutes with gentle rocking.Then this solution was centrifuged 10,000 g for 13 minutes, thesupernatant collected and treated with DNase/RNase preparation (1 mg/mlof each enzyme in total 1 ml stock solution) to make the sample 25 ugDNase and 25 ug RNase per 5 ml, incubated at 37° C. for one hour, thencentrifuged 5000 g for 10 minutes, the supernatant collected, filteredwith a 0.22 micron Millipore™ (Millipore Inc., Bedford, Mass.) filter.Samples of 5 ml each were applied to a HiPrep™ 26/60 Sephacryl™ S-200 HRcolumn (GE Healthcare Bio-Sciences Corp., Piscataway, N.J.) at flow rate1.0 ml/min in 0.05 M phosphate, 0.15 M NaCl buffered saline, pH 7.8.Fractions 30-33 (total 7 cc per run) were collected and concentratedusing Millipore™ spin concentrators (nominal molecular weight limit 100kDa). The resulting samples were diluted 1:1 with Protein A IgG Bindingbuffer (0.02 M Na citrate, 0.1M phosphate buffer, pH 7.4, PierceChemical Co., Rockford, Ill.), and applied to a Protein A column (5 mlProtein A/column) (Pierce). The column was then washed with bindingbuffer (10× column volume and repeated until an absorption reading ofzero was observed), and then immunoglobulin was eluted with Immunopure™IgG elution buffer (0.02 M Na citrate, 0.1M NaCl, pH 2.5, Pierce) at 1ml/min. Fractions were collected, and samples were brought to a finalconcentration of 0.05 M phosphate, 0.15 M NaCl buffered saline, pH 7.4,using Millipore concentrators according to the manufacturer'srecommendations by performing about 2-3 cycles of concentrating and thenadding buffer. Protein concentrations were assayed using the BCAtechnique (Pierce) and then samples were stored at −20° C.

SDS-PAGE and Protein Identification

Samples were diluted to 1:5 in HES buffer (10 mM HEPES, 10 mM EDTA, 250mM sucrose), and then diluted to 1:1 with reducing sample buffer(Pierce). The samples were then boiled for 10 minutes, and SDS-PAGE (8%)was performed to obtain separated immunoglobulin heavy (H) and light (L)chains. The gels were stained using Imperial Protein Stain (Pierce) inorder to cut out the H and L chain bands. Proteomic analysis of the Hand L chain bands was performed by ProtTech (Norristown, Pa.) usingproprietary techniques and mass spectrometery. N-terminal amino acidsequencing of H and L chains was obtained by preparing H and L chains bySDS-PAGE, transferring H and L chains to polyvinylidene fluoride (PVDF)membranes, staining the membranes with MemCode™ Reversible Protein StainKit for PVDF membrane (Pierce), cutting out the H and L bands, andsending the bands to Alphalyze, Inc. (Palo Alto, Calif.) to perform thesequencing.

The placental tissue lysate samples from 11 donors were examined underreducing and non-reducing conditions using SDS-PAGE and showed anelectrophoretic migration pattern suggesting, respectively, the intactform (non-reducing) of the immunoglobulin and the separated (reducing) Hand L chains of immunoglobulin G. Since these results suggested a uniqueplacental immunoglobulin G, henceforth this protein was identified asIgX. The bands corresponding to the H and L chains were sent for proteinanalysis and indeed showed the presence of H and L chains, but also someimpurities.

In order to enhance the purification of these bands for proteinanalysis, the placental tissue lysates from four donors (RA, BC, MP, andKS) were treated under non-reducing conditions with gel filtrationfollowed by protein A purification and then tested under reducingcondition with SDS-PAGE. The H and L bands were sent for proteinidentification and cross matching with other protein sequences in theprotein data bank and were found to have nearly identical sequences witheach other and with an IgG1 H chain identified as GenBank accessionnumber AAH90938.1 (SEQ ID NO:3, Table 1). The sequences were found to benearly identical at residues 1-19, 133-157, 298-311, 325-343, 340-347,368-383, 395-432, as shown by the alignments in Table 1.

The H bands and L bands from the SDS-PAGE gel were transferred to PVDF,stained, excised and sent for N-terminal amino acid sequencing. Theseresults showed that sample KS had 70-80% credibility of having theAAH90938.1 N-terminal sequence EVQLVE (SEQ ID NO: 110), and the aminoterminal sequence for sample BC H chain was also highly homologous toEVQLVE (SEQ ID NO: 110). Interestingly, the N-terminal sequence forsample BC H chain was identified as DVQLLE (SEQ ID NO: 111) by proteinanalysis using mass spectrometry. These N-terminal amino acid sequenceswere nearly identical to the amino acid sequence of AAH90938.1 atresidues 19-24. Therefore, the preceding (upstream) residues werebelieved according to non-limiting theory to represent a signal sequencefor the H chain.

TABLE 1IgX H chain partial sequences from four different placental samples (SEQ ID NOS: 3, 112, 113, 114, 115):AAH90938.1mefglswvfl vailkgvqce vqlvesgggl vqpgrslrls ctssgftfgd yamnwvrqap gkglewvgfi rskpyggtte yaaslkgrft vsrddsksiaKS--------------------e vklvesgggl vqpgrslr--------------------------------------------------------------------MP--------------------e vklvesgggl vqpgrslr--------------------------------------------------------------------BC--------------------d vqllesgggl vqpggslr--------------------------------------------------------------------RA--------------------e vqlvesgggl vqpgrslr--------------------------------------------------------------------AAH90938.1ylqmnslkte dtalyyctrs lrgvqqpldy wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv swnsgaltsg vhtfpavlqsKS------------------------------------gtlvtvs sastkgpsvf plapssk----- -----------------------------------------MP------------------------------------gtlvtvs sastkgpsvf plapssk-----------------------------------------------BC-----------------------------------              gpsvf plapssk-----------------------------------------------RA-----------------------------------              gpsvf plapssk--------- -------------------------------------AAH90938.1sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkkve pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfnKS------------------------------------------------------------------------- ----- ------- ------ ----------- fnMP-----------------------------------------------------------------------------------------------------------fnBC-----------------------------------------------------------------------------------------------------------fnRA------------------------------------------------------------------------------------------------------ ----fnAAH90938.1wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti skakggprep qvytlppsrd eltknqvslt clvkgfypsdKSwyvdgvevhn ak------------- vvsvl tvlhqdwlng keyk-------       ------------ep qvytlppsr-----------------gfypsdMPwyvdgvevhn ak--------------vvsvl tvlhqdwlng keyk-------alpapiek-----------ep qvytlppsre emtk-----------gfypsdBCwyvdgvevhn ak--------------vvsvl tvlhqdwlng keyk-------alpapiek-----------ep qvytlppsre emtk-----------gfypsdRAwyvdgvevhn ak--------------vvsyl yvlhqdwlng keyk-------alpapiek-----------ep qvytlppsrd eltk-----------gfypsdAAH90938.1iavewesngq pennykttpp vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg kKSiavewesngq pennykttpp vldsdgsffl ysk------------------------------------------MPiavewesngq pennykttpp vldsdgsffl ysk------------------------------------------BCiavewesngq pennykttpp vldsdgsffl ysk------------------------------------------RAiavewesngq pennykttpp vldsdgsffl ysk------------------------------------------SEQ ID NO: 3: (Acc. No. AAH90938.1 GI:60551126)  1 MEFGLSWVFL VAILKGVQCE VQLVESGGGL VQPGRSLRLS CTSSGFTFGD YAMNWVRQAP 61 GKGLEWVGFI RSKPYGGTTE YAASLKGRFT VSRDDSKSIA YLQMNSLKTE DTALYYCTRS121 LRGVQGPLDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV181 SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE241 PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN301 WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI361 SKAKGQPREP QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP421 VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K(Strausberg et al., 2002 Proc. Nat. Acad. Sci. 99:16899)

Example 2 IgX Induces Cytokine Production

In order to examine the biological activity of IgX, cytokine productionfrom human peripheral whole blood samples incubated with IgX wasmeasured. Peripheral whole blood samples from three different donors(#66, 67, and 85) were obtained with appropriate informed consent andincubated with concentrations of IgX ranging from 0 to 200 ug/ml.

Cytokine Analysis

Protein A purified IgX was prepared from placental sample BC, brought to0.05 M phosphate, 0.15 M NaCl buffer, pH 7.4, treated with an endotoxinaffinity column (ActiClean Etox™ from Sterogene, Carlsbad, Calif.)according to the supplier's instructions, and then sent to IBTBioservices (Gaithersburg, Md.) for human peripheral blood testing. Thissample was found to have 1 EU/ml of endotoxin per 10 ug/ml of sample.All peripheral blood cells were suspended in RPMI-1640 media. Variousconcentrations of IgX were incubated with peripheral blood cells fromdonor D66 (50 y/o female) for 18 hours, and then the samples werestimulated with 1 ug/ml of PHA, 50 ng/ml of LPS, or 10 ng/ml of IFNγ(200 U/ml) for four hours. Samples of culture supernatant fluids weretested in duplicate for the presence of IL-2, IL-4, IL-6, IL-10, IL-12,and TNFα using a Meso Scale Discovery (MSD, Gaithersburg, Md.) panel.

Production of the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, and TNFα wasdetermined for peripheral blood cells following exposure to IgX. Cellsfrom donor #66 were sensitive to lower doses of IgX compared to theother donors, and these cells showed a significant increase in IL-6production and, to a lesser degree, an increase in TNFα production at 10ug/ml IgX (FIGS. 1 and 2). Cells from the other donors showed asignificant increase in IL-6 and, to a lesser degree, an increase inTNFα, at higher concentrations of IgX ranging from 100 to 200 ug/ml(FIGS. 3, 5 and 6). Cells from donor #85 also had a slight increase inIL-10 production at 200 ug/ml IgX (FIG. 4). There was no significantincrease in the other cytokines measured (i.e., IL-2, IL-4 and IL-12).IgX did not appear to have an immunosuppressive effect on peripheralblood cells when incubated in the presence of LPS, PHA or IFNγ.

Example 3 Active Fragments of IgX

In order to determine if the immunomodulating potential of IgX lieswithin the Vh hypervariable region of AAH90938.1 (SEQ ID NO:3), peptidescomprising amino acid sequences that corresponded to the thirdhypervariable region and third framework region were prepared. PeptideX1had the amino acid sequence AEDTAVYYCAR (SEQ ID NO:1) of the H chain ofsample BC, which shared 73% homology with AAH90938.1 at residues109-119. Peptide X2 had the amino acid sequence KSIAYLQMNSLK (SEQ IDNO:2) that corresponded to residues 97-108 of the H chain of AAH90938.1.

Peptide Preparation

PeptideX1 and PeptideX2 were synthesized by Genscript, Inc. (Piscataway,N.J.) and solubilized by adding 50 ul of 10% acetic acid to 2 mg ofsample. Then 450 ul of 0.1 M Tris buffer was added to each sample toprovide a total volume of 500 ul at pH 7. Genscript also preparedconjugates of peptideX2-FITC (fluorescein isothiocyanate) with thefluoroprobe attached to the COOH end of peptideX2. 2 mg peptideX2-FITCwas solubilized by adding 20 ul of 5% acetic acid solution, and then 980ul of 0.01 M phosphate buffered saline, pH 6, was slowly added.

Cytokine Analysis

PeptideX1 and PeptideX2 were incubated with peripheral blood cells fromdonor D67 (30 y/o female) as described above and tested in duplicate forIL-6 activity. Another set of experiments examined peptideX2 and IgX,both prepared in 0.1 M Tris buffer, pH 7, and incubated with peripheralblood cells from donor D68 (27 y/o female). IgX was made endotoxin freeby adding 4 ml of IgX 0.1 M Tris, pH 8.8, to a Strong Anion ExchangeSpin Column (Pierce), centrifuging at 200×g for 5 minutes, washing twicewith sterile 0.1 M Tris, pH 8.8, eluting from the membrane with twowashes with sterile 0.1 M Tris, pH 7.2, and then concentrating thesample. All peripheral blood cells were suspended in RPMI-1640 media.

Flow Cytometry

Peripheral blood samples in EDTA were obtained with appropriate informedconsent. 4 ml of ACK lysis buffer was added for every 4 ml of bloodsample and allowed to mix in a 50 ml conical tube for 3-5 minutes atroom temperature. The sample was centrifuged at 350 g for 5 minutes andthe supernatant was discarded. Next, 2 ml of ACK lysis buffer was addedand gently mixed with the cells prior to incubating the sample for 3-5minutes. The sample was then centrifuged at 350×g for 5 minutes, and thesupernatant was discarded. Next, 500 ul of stock Fc blocking solution(consisting of 40 ul of Fc blocking solution from eBiosciences, Inc.(San Diego, Calif.) plus 1 ml of 0.01 M phosphate buffered saline and 3%BSA) was added to the cell pellet and mixed gently for 10 minutes atroom temperature. 100 ul aliquots were prepared with variousconcentrations of peptideX2-FITC and 1 ul of anti-CD markers (CD5-APC,CD14-PE, CD181-PE-Cy5, CD56-PE-Cy5) for each test sample and incubatedfor 15 min at room temperature. The reactions were stopped by adding 900ul of cold PBS followed by centrifugation at 9000×g for 3 minutes.Supernatants were decanted, and the cold PBS wash with gentle vortexingwas repeated with centrifugation following again, at 5800 rpm for 3minutes. The supernatant was decanted again. The washing process wasrepeated a total of three times. After washing, 1 ml of 0.1% TritonX-100 in PBS was added and gently mixed with the cells. The cells wereincubated at room temperature for 10 minutes before centrifuging at 5800rpm for 3 min minutes and decanting the supernatant. Next, 500 ul of 1%paraformaldehyde in PBS was added to the cells and mixed gently. Thecells were incubated for 10 min and then stored in the dark at 4° C. forup to 3 days. Results were acquired using a FACS-CALIBUR BD 4 Color flowcytometer (Becton Dickinson & Co., Rockville, Md.) according to themanufacturer's instructions. A macrophage cell line (THP-1) treated with2 mg LPS incubated for 12 hours and without LPS was also examined.Following the 12 hour incubation with or without LPS, peptideX2-FITC wasadded at various concentrations and incubated for 15 minutes. The cellswere then treated as described above and analyzed using flow cytometry.

Using cells from donor #67, peptideX2 induced a significantly greaterIL-6 production compared to peptideX1 (FIG. 7). PeptideX2 was testedagain using cells from a different donor (#85), and again it was foundto induce enhanced production of IL-6 and, to a lesser degree, TNFα andIL-10 (FIGS. 8 and 9).

In order to study the cell binding of pepideX2, the fluorescentconjugate peptideX2-FITC was prepared. However, it was important todetermine whether or not conjugating the FITC to the COOH end ofpeptideX2 would alter its biological activity. Therefore, keyhole limpethemocyanin (KLH) was conjugated to peptideX2 at its COOH end, and thebiological activity was tested and found to be intact. In fact,KLH-peptideX2 had a significant positive impact on the production ofIL-6 (FIG. 10). PeptideX2-FITC and antibodies for cell markers wereincubated with human peripheral blood cells and prepared for flowcytometry. Surprisingly, PeptideX2-FITC bound to a subset of neutrophilsbut did not bind to NK cells, T cells, monocytes, and B cells atconcentrations less than 20 ug/ml. Some nonspecific binding was observedat concentrations greater than 20 ug/ml. PeptideX2 was also incubated inthe presence or absence of LPS in a monocyte cell line but without anybinding.

Example 4 Amino Acid Sequence Homology to PeptideX2

In order to determine if there were any reported peptides having a highpercentage of amino acid sequence identity to the 12 amino acid sequenceof PeptideX2 [SEQ ID NO:2], a BLAST search of the NCBI amino acidsequence database was performed. Table 2 is a summary of the results,which revealed 100 polypeptides having sequences that included the 12contiguous amino acids of SEQ ID NO:2. All of the amino acid sequencesidentified were human immunoglobulin G1 Vh domains of 67 amino acids inlength or longer, derived from IgG1(K) immunoglobulins, and each oneincluded, at varying positions within the sequence, the dodecamericPeptideX2 sequence, KSIALYQMNSLK (SEQ ID NO:2).

Homologues of the dodecameric PeptideX2 sequence (SEQ ID NO:2) were alsoidentified as sequence variants that were present in murine and ratimmunoglobulin heavy chain amino acid sequences in the database, wheresuch variants differed from SEQ ID NO:2 at no more than 5, 4, 3, 2 or 1positions within the amino acid dodecamer, such differences beingpresent as substitutions, deletions or insertions. Relative to the humansequence in which SEQ ID NO:2 was identified, a greater number ofvariants was found among murine sequences than among rat sequences,suggesting evolutionary conservation of SEQ ID NO:2. Certain of thepresently contemplated embodiments, however, may employ at least 1, 2,3, 4 or 5 of the presently described SEQ ID NO:2 variants throughgeneration of variant PeptideX2 structures that comprise the amino acidsequence of general formula:

K-X1-X2-X3-YLQM-X4-X5-LK as set forth in SEQ ID NO:106,

wherein X1 is selected from S and N, X2 is selected from I, T, S, M, Rand N, X3 is selected from A, L, V and Q, X4 is selected from N, D, S, Tand A, and X5 is selected from S, T and N.

TABLE 2 Sequences producing significant alignments with PeptideX2 SEQ IDMax Total Query E NO: Accession Description score score coverage value 5ADW08230.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 6 ADW08228.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 7 ADW08227.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 8ADX89690.1 immunoglobulin epsilon heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 9 ADX89674.1 immunoglobulin epsilon heavychain variable region [Homo sapiens] 41.8 41.8 100% 0.012 10 ADX65711.1immunoglobulin variable region [Homo sapiens] 41.8 41.8 100% 0.012 11ADX65652.1 immunoglobulin variable region [Homo sapiens] 41.8 41.8 100%0.012 12 ADX65553.1 immunoglobulin variable region [Homo sapiens] 41.841.8 100% 0.012 13 ADX65550.1 immunoglobulin variable region [Homosapiens] >gb|ADX65551.1| 41.8 41.8 100% 0.012 immunoglobulin variableregion [Homo sapiens] 14 ADX65549.1 immunoglobulin variable region [Homosapiens] 41.8 41.8 100% 0.012 15 ADX65548.1 immunoglobulin variableregion [Homo sapiens] 41.8 41.8 100% 0.012 16 ADX65545.1 immunoglobulinvariable region [Homo sapiens] 41.8 41.8 100% 0.012 17 ADX65526.1immunoglobulin variable region [Homo sapiens] >gb|ADX65541.1| 41.8 41.8100% 0.012 immunoglobulin variable region [Homo sapiens] >gb|ADX65542.1|immunoglobulin variable region [Homo sapiens] 18 ADU57684.1anti-vaccinia virus immunoglobulin heavy chain variable region 41.8 41.8100% 0.012 [Homo sapiens] 19 ADQ01609.1 immunoglobulin heavy chainvariable region 41.8 41.8 100% 0.012 [Homo sapiens] >gb|ADQ01610.1|immunoglobulin heavy chain variable region [Homosapiens] >gb|ADQ01662.1| immunoglobulin heavy chain variable region[Homo sapiens] >gb|ADQ01681.1| immunoglobulin heavy chain variableregion [Homo sapiens] >gb|ADQ01683.1| immunoglobulin heavy chainvariable region [Homo sapiens] 20 ADM44271.1 immunoglobulin gamma 1heavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 21ADM43803.1 immunoglobulin gamma 3 heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 22 ADD14319.1 immunoglobulin heavy chain[Homo sapiens] 41.8 41.8 100% 0.012 23 ADD14256.1 immunoglobulin heavychain [Homo sapiens] 41.8 41.8 100% 0.012 24 BAI51461.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 25BAI51432.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 26 BAI52610.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 27 BAI52607.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 28BAI52605.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 29 BAI52598.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 30 BAI52406.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 31BAI52390.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 32 BAI52365.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 33 BAI52322.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 34BAI52220.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 35 BAI52189.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 36 BAI52163.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 37BAI52156.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 38 BAI52150.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 39 BAI52017.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 40BAI52008.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 41 BAI51980.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 42 BAI51969.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 43BAI51874.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 44 BAI51746.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 45 BAI51738.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 46BAI51639.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 47 BAI51627.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 48 BAI51598.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 49BAI51315.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 50 BAI50966.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 51 ACR16225.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 52ACR16214.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 53 ACR16203.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 54 ACT68811.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 55ACE75034.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 56 ACN43624.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 57 CAR62757.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 58CAP78944.1 immunoglobulin heavy chain variable region 41.8 41.8 100%0.012 [Homo sapiens] >gb|ADQ01474.1| immunoglobulin heavy chain variableregion [Homo sapiens] >gb|ADQ01475.1| immunoglobulin heavy chainvariable region [Homo sapiens] >gb|ADQ01482.1| immunoglobulin heavychain variable region [Homo sapiens] >gb|ADQ01514.1| immunoglobulinheavy chain variable region [Homo sapiens] >gb|ADQ01549.1|immunoglobulin heavy chain variable region [Homosapiens] >gb|ADQ01563.1| immunoglobulin heavy chain variable region[Homo sapiens] 59 CAP78943.1 immunoglobulin heavy chain variable region41.8 41.8 100% 0.012 [Homo sapiens] >gb|ADQ01245.1| immunoglobulin heavychain variable region [Homo sapiens] >gb|ADQ01263.1| immunoglobulinheavy chain variable region [Homo sapiens] >gb|ADQ01299.1|immunoglobulin heavy chain variable region [Homosapiens] >gb|ADQ01358.1| immunoglobulin heavy chain variable region[Homo sapiens] >gb|ADQ01505.1| immunoglobulin heavy chain variableregion [Homo sapiens] >gb|ADQ01599.1| immunoglobulin heavy chainvariable region [Homo sapiens] 60 ABW80076.1 immunoglobulin heavy chainvariable region [Homo sapiens] 41.8 41.8 100% 0.012 61 ABW79987.1immunoglobulin heavy chain variable region [Homo sapiens] 41.8 41.8 100%0.012 62 ABW79941.1 immunoglobulin heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 63 ABP98602.1 immunoglobulin heavy chainvariable region [Homo sapiens] 41.8 41.8 100% 0.012 64 ABP98457.1immunoglobulin heavy chain variable region [Homo sapiens] 41.8 41.8 100%0.012 65 ABP98369.1 immunoglobulin heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 66 ABP98398.1 immunoglobulin heavy chainvariable region [Homo sapiens] 41.8 41.8 100% 0.012 67 ABP98334.1immunoglobulin heavy chain variable region [Homo sapiens] 41.8 41.8 100%0.012 68 ABP98180.1 immunoglobulin heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 69 ABP98113.1 immunoglobulin heavy chainvariable region [Homo sapiens] 41.8 41.8 100% 0.012 70 ABP98000.1immunoglobulin heavy chain variable region [Homo sapiens] 41.8 41.8 100%0.012 71 ABP98003.1 immunoglobulin heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 72 ABP97942.1 immunoglobulin heavy chainvariable region [Homo sapiens] 41.8 41.8 100% 0.012 73 ABP97768.1immunoglobulin heavy chain variable region [Homo sapiens] 41.8 41.8 100%0.012 74 ABP97575.1 immunoglobulin heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 75 ABP97570.1 immunoglobulin heavy chainvariable region [Homo sapiens] 41.8 41.8 100% 0.012 76 ABV70953.1immunoglobulin heavy chain variable region [Homo sapiens] 41.8 41.8 100%0.012 77 ABM67236.1 immunoglobulin heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 78 CAK50728.1 immunoglobulin A heavy chainvariable region [Homo sapiens] 41.8 41.8 100% 0.012 79 ABI35565.1immunoglobulin heavy chain variable region [Homo sapiens] 41.8 41.8 100%0.012 80 ABM53261.1 immunoglobulin heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 81 EAW82007.1 hCG2029223 [Homo sapiens]41.8 41.8 100% 0.012 82 ABK81362.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 83 ABK81417.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 84ABJ97553.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 85 ABI74220.1 immunoglobulin heavy chain variableregion 41.8 41.8 100% 0.012 [Homo sapiens] >gb|ABI74221.1|immunoglobulin heavy chain variable region [Homo sapiens] 86 ABI74341.1immunoglobulin heavy chain variable region [Homo sapiens] 41.8 41.8 100%0.012 87 ABI74230.1 immunoglobulin heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 88 ABG38442.1 immunglobulin heavy chainvariable region [Homo sapiens] 41.8 41.8 100% 0.012 89 AAS85995.1immunoglobulin heavy chain [Homo sapiens] 41.8 41.8 100% 0.012 90AAS86095.1 immunoglobulin heavy chain [Homo sapiens] 41.8 41.8 100%0.012 91 AAQ87970.1 immunoglobulin E heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 92 CAE45439.1 immunoglobulin heavy chainvariable region [Homo sapiens] 41.8 41.8 100% 0.012 93 CAC10788.1immunoglobulin heavy chain variable region [Homo sapiens] 41.8 41.8 100%0.012 94 CAD19295.1 immunoglobulin heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012 95 CAD44709.1 immunoglobulin heavy chainvariable region [Homo sapiens] 41.8 41.8 100% 0.012 96 CAA12632.1 Igheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 97CAD60291.1 immunoglobulin heavy chain variable region [Homo sapiens]41.8 41.8 100% 0.012 98 CAC94369.1 immunoglobulin heavy chain variableregion [Homo sapiens] 41.8 41.8 100% 0.012 99 CAD60306.1 immunoglobulinheavy chain variable region [Homo sapiens] 41.8 41.8 100% 0.012 100BAC02049.1 immunoglobulin heavy chain VHDJ region [Homo sapiens] 41.841.8 100% 0.012 101 BAC02301.1 immunoglobulin heavy chain VHDJ region[Homo sapiens] 41.8 41.8 100% 0.012 102 CAC10773.1 immunoglobulin heavychain variable region [Homo sapiens] 41.8 41.8 100% 0.012 103 BAC02007.1immunoglobulin heavy chain VHDJ region [Homo sapiens] 41.8 41.8 100%0.012 104 CAD60290.1 immunoglobulin heavy chain variable region [Homosapiens] 41.8 41.8 100% 0.012

Example 5 PeptideX2 Inhibited the Growth of E. coli

The results demonstrated in the previous Examples suggested thatneutrophils may play a significant role in fetal-maternal allograftacceptance, and that IgX may be a significant immunomodulating moleculethat exerted its immunomodulatory effect through the presence within theIgX heavy chain Vh domain of the PeptideX2 structure. To test thishypothesis, human peripheral white cells were incubated with E. coli inthe presence and absence of PeptideX2.

Human peripheral blood leukocytes (WBCs) were obtained from healthydonors with informed consent. Three test groups were inoculated with E.coli and incubated for 10 hours. The three groups were E. coli withserum alone, E. coli with WBCs, and E. coli with WBCs with PeptideX2.Samples were taken at 0, 2, 4, 6 and 10 hours and plated for colonyforming units (CFUs). The number of E. coli present in the serumincreased modestly over time, while the number of E. coli present in theWBC group showed only a slight increase. In contrast, the E. colipresent in the WBC experimental group that was incubated with PeptideX2group exhibited a rapid rise in CFUs at 10 hours (FIG. 11). In aseparate but similar experiment, nine test groups (E. coli with serumalone, E. coli with WBCs, and E. coli with WBCs with PeptideX2 at eachof seven indicated concentrations, see FIG. 12) were incubated over atime course of three hours, with quantification of bacterial CFUperformed at times 0, 1.5 and 3 hours (FIG. 12). These results suggestedthat PeptideX2 suppressed the innate immune response that was mediatedby WBC in the absence of PeptideX2, and that PeptideX2 may down regulatethe adaptive immune response.

Example 6 Amino Acid Sequence Alignment of PeptideX2 Against PIFPeptides

Pre-implantation Factor (PIF) peptides are naturally occurring peptidesthat have been shown to promote suppression of the maternal immuneresponse during pregnancy and may be important for embryo implantation.Several PIF peptides have been identified (see, e.g., PCT ApplicationPublication Nos. WO 2003/004601 and WO 2005/040196). In order to confirmthat PeptideX2 was not a known PIF peptide, the amino acid sequences of8 PIF peptides (sequences 1-8 in Table 3) were compared to the aminoacid sequence of PeptideX2. As shown in Table 3 below, none of the PIFsequences share any sequence identity with PeptideX2.

TABLE 3 PIF and PeptideX2 amino acid sequence comparison SequenceAmino acid residues sequence #1 G K R I K G T sequence #2 V L G K R I KG T sequence #3 I E V L G K R I K G T sequence #4 I D V L G K R I K G Tsequence #5 I R V L G K R I K G T sequence #6 I E V T G K R I K G Tsequence #7 I D V T G K R I K G T sequence #8 I R V T G K R I K G TpeptideX2 K S I A Y L Q M N S L K HOMOLOGY no no no no no no no no no nono no

Example 7 In Vitro Allograft Cell Proliferation Assays with PeptideX2

A modified mixed lymphocyte reaction (MMLR) is used to determine if theimmunomodulating properties of PeptideX2 are sufficient to suppress cellproliferation in an in vitro allograft model. Mixed lymphocyte reactionsare used to test the compatibility of lymphocytes from two individuals.One set of lymphocytes is irradiated or treated with mitomycin C so thatthey cannot respond or proliferate in response to a stimulus, and theother set of lymphocytes are responder cells which can differentiateinto effector cells and proliferate if they are alloreactive to thefirst set of lymphocytes. The MMLR includes test groups featuring theaddition of PeptideX2 to determine its suppressive effect on cellproliferation.

Cultures of peripheral white blood cells of eight human subjectsobtained with appropriate informed consent are analyzed using one-wayand two-way MMLR. Cells are cultured with 250 ug/ml PeptideX2 in one-wayreactions using mitomicyin C. Two-way MMLR are also performed using 250ug/ml PeptideX2. Cell proliferation is measured using a BrdUcolorimetric assay according to the manufacturer's instructions (RocheApplied Science Product #11647229001). Inhibition of cellularproliferation by at least 50% indicates that a concentration ofPeptideX2 is sufficient to suppress cell proliferation.

Optimum dosing of PeptideX2 is determined by measuring the degree ofinhibition of cellular proliferation using various concentrations ofPeptideX2. The concentrations include 25, 50, 100, 250 and 500 μg/ml.The dose of PeptideX2 that results in the greatest alteration (e.g.,statistically significant decrease in cytoproliferation) in MMLR assaysis regarded as the optimum dose for suppressing cellular proliferation.

Example 8 In Vitro Xenograft Cell Proliferation Assays with PeptideX2

Similarly, MMLR assays are used to determine if PeptideX2 may be usefulin suppressing cellular proliferation as an in vitro xenograft model.The MMLR assays are performed as described above, however, mixtures ofhuman and non-human leukocytes are used to determine the optimum dose orconcentration of PeptideX2 for suppressing cellular proliferation in axenograft model. The non-human leukocytes are obtained from pigs, ratsand monkeys.

Example 9 Generation of PeptideX2 Variants

Variants of PeptideX2 are generated by introducing amino acidsubstitutions at different positions in the PeptideX2 amino acidsequence in order to determine if such variations enhance the biologicalactivity of PeptideX2. Preferred variants are polypeptides of 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31amino acids that comprise at least one amino acid sequence as set forthin SEQ ID NO:106; in embodiments where the peptide comprises apolypeptide of SEQ ID NO:106 that differs in amino acid sequence fromSEQ ID NO:2, polypeptides of no more than 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 amino acids, or ofno more than 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61-70, 71-80 or81-90 amino acids may be used. The modified peptides are tested ascandidate competitive inhibitors of PeptideX2-FITC (described above)binding to neutrophils, using flow cytometry in order to compare theiractivity. Competitive cell-binding assays are performed by flowcytometry as described above, and cytokine induction profiles are alsogenerated as described above, in order to compare the modified PeptideX2sequences with unmodified PeptideX2. Peptides exhibiting significantactivity are selected for use in MMLR and in vivo transplant modelshaving relevance to organ transplantation and allograft rejection asdiscussed herein.

In flow cytometry-based competitive binding assays, cells are incubatedwith either PeptideX2-FITC or PeptideX2-FITC conjugated to BSA and thePeptideX2 sequence variant, to determine if the variant competes withPeptideX2. The competitive binding assays are used in addition to thecytokine profile results in order to determine candidate PeptideX2variants for use in medical treatments such as transplantation.

Example 10 In Vivo Transplant Model

In order to further examine the potential role of PeptideX2, or asuitable variant, established in vivo allograft and xenograft models areutilized. For a xenograft model, human tissue is transplanted into ananimal, such as a rodent or primate. Control animals are not treatedwith PeptideX2, and test animals are treated with either PeptideX2 or astandard immunosuppressive agent, such as anti-IL-2Rα. The rate of graftacceptance and/or rejection between the treatment groups is compared.

Example 11 Immune Status Profile of PeptideX2-Induced Immunomodulation

Immune status profiling is performed by determining the effects, onperipheral blood white blood cells, of exposure to a herein providedpolypeptide that comprises the PeptideX2 sequence (SEQ ID NO:2) or thatcomprises the PeptideX2 variant sequence (SEQ ID NO:106) as describedherein. MLR and MMLR assays are performed as described above, and theeffects of PeptideX2 (SEQ ID NO:2) sequence-containing polypeptides orPeptideX2 variant (SEQ ID NO:106) sequence-containing polypeptides onMLR and MMLR are determined by measuring cellular proliferation and alsoby characterizing supernatant fluids for released cytokines using artaccepted assay methodologies (e.g., Ready-Set-Go™ ELISA kit fromeBiosciences, San Diego, Calif.; or with immunoassay kits as are readilyavailable from R & D Systems, Minneapolis, Minn., or from BDBiosciences, San Jose, Calif.; or using other well known methodologiesfor detecting and quantifying cytokines). The effects of PeptideX2 orPeptideX2 variant sequence-containing polypeptides are also assessed onin vitro peripheral blood leukocyte (PBL) cultures following stimulationwith T cell mitogens (e.g., PHA, or costimulatory antibody combinationssuch as anti-CD3/anti-CD28, etc.) or with B cell mitogens (e.g., LPS, orcostimulatory antibody combinations, etc.) or with other established PBLsubpopulation-specific or non-specific stimulatory protocols.

Following in vitro stimulation in controlled experimental groups thatinclude such treatments in the presence and absence of a PeptideX2 orPeptideX2 variant sequence-containing polypeptide as provided herein,culture fluids are separated from cells and tested for the presence ofone or more members of a panel of immunologically relevant cytokines.The panel includes: IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, and TNF-α(detection of these cytokines is also described above) and also includesone or more of IL-1p, IL-5, IL-8, IL-13, IL-17, IL-22, CCL2, CCL3, CCL4,CCL5, CCL11, CXCL5, CXCL11, TGFβ, IFNγ, basic FGF, GCSF, GMCSF, VEGF,EGF, and HGF.

Peripheral blood white cells containing peripheral blood leukocytes arealso sorted using fluorescence activated cell sorting (FACS) on thebasis of positive staining with labeled PeptideX2 or IgX, and thepositively selected cells are cultured in culture medium alone or inmedium supplemented with PeptideX2, IgX and/or other neutrophil-inducingagents. At suitable timepoints medium aliquots are collected andseparated from cells, and the supernatant fluids are tested for thepresence of one or more of the members of the panel of cytokines asdescribed above, in order to profile the cytokine-elaboration activitystatus of PeptideX2-expressing cells.

Example 12 PeptideX2 Interaction with Human TLR2 and TLR4 and withMurine TLR2 and TLR4

PeptideX2 (200 μg/mL) was tested for its effects on human TLR-drivenNF-κB activation in HEK293 cell lines transfected with human TLR2, 3, 4,5, 7, 8 or 9 (InvivoGen, San Diego, Calif.). HEK293 cells (50,000 to75,000 cells/well) expressing the indicated TLR were plated in wells ofa 96-well plate (200 μl total volume) containing either with noadditions, PeptideX2 (in 20 μl buffer), or buffer only (20 μl) as avehicle control. All cells contained a reporter construct having codingsequence for Secreted Embryonic Alkaline Phosphatase (SEAP) under thecontrol of a promoter inducible by the transcription of NF-kB, and wereincubated in media containing a detectable SEAP expression indicator(InvivoGen, San Diego, Calif.). After 16-20 hours incubation at 37° C.,the optical density of culture supernatants was read at 650 nm on aMolecular Devices SpectraMax™ 340PC absorbance detector. Representativeresults are shown in FIG. 13, in which evidence for PeptideX2 activationof NF-κB via TLR2 and TLR4 can be seen.

The relative potency of PeptideX2 induction of human TLR-mediated NF-κBactivation was compared to the effects of known PAMP ligands that haveactivity as agonists for each of the human TLRs TLR2, TLR3, TLR4, TLR5,TLR7, TLR8 and TLR9, using the NF-κB-driven SEAP reporter assay(InvivoGen, San Diego, Calif.). The known TLR agonists were as follows:for TLR2, heat killed Listeria monocytogenes (HKLM) at 10⁸ cells/ml; forTLR3, poly(I:C) at 1 ug/ml; for TLR4, E. coli K12 LPS at 100 ng/ml; forTLR5, S. typhimurium flagellin at 100 ng/ml; for TLR7, CL097 at 1 ug/ml;for TLR8, CL075 at 1 ug/ml; for TLR9, CpG ODN 2006 at 100 ng/ml.

Representative results are shown in FIG. 14; NF-κB-negative control celllines were also tested and produced negative results (not shown). InFIG. 14, PeptideX2 elicited a TLR2-mediated response at a level that was14% of the response by the TLR2-transfected cells to HKLM; and aTLR4-mediated response at a level that was 10% of the response by theTLR4-transfectants to E. coli K12 LPS. For the human TLR2 transfectants,responses to varying concentrations of PeptideX2 were measured in theNF-κB-driven SEAP reporter assay (InvivoGen, San Diego, Calif.).Representative results are shown in FIG. 15; in this assay the positivecontrol TLR2 agonist (HKLM) elicited an OD (650 nm) reading of 2.5, andnegative control cultures released no detectable SEAP. PeptideX2 (200μg/mL) was also tested for its effects on murine TLR-driven NF-κBactivation in HEK293 cell lines transfected with murine TLR2, 3, 4, 5,7, 8 or 9 (InvivoGen, San Diego, Calif.) essentially as described above.As shown in FIG. 18, the results again showed induction by PeptideX2 ofNF-κB-driven SEAP expression, acting through TLR2 and TLR4 but notthrough the other TLRs tested. Using the murine TLR transfectants inthis experiment, PeptideX2 exhibited greater potency in activating viamurine TLR4 relative to murine TLR2 (FIG. 18).

Example 13 Early-Phase Effects of PeptideX2 on Leukocyte Inhibition ofBacterial Growth and Inflammatory Cytokine Release

Staphylococcus aureus bacteria were incubated with human peripheralblood white cells (WBC) in the absence or presence of PeptideX2 (0, 10,and 100 μg/ml), and the effects on S. aureus growth were assessed byserial dilutions, microbiological plating and determination by countingcolony forming units (CFU) in culture samples withdrawn after incubationtimes of 0, 1.5 and 3 hours. The WBC-to-Staph ratio was 100:1, using WBCat 4×10⁶/ml and Staph 4×10⁴ CFU/ml.

As shown in FIG. 16, no S. aureus growth was detected when WBC werepresent for up to three hours of incubation regardless of the presenceor absence of PeptideX2, apparently due to WBC killing of the bacteria.Consistent with WBC-mediated bacteriocidal activity, no inhibition ofCFU formation was observed when S. aureus was cultured in the presenceof serum alone with no WBC.

Supernatant fluids from the same experimental cultures were also testedfor TNFα production by WBC during the short-term incubations with S.aureus. TNFα was quantified by enzyme-linked immunosorbent assay (ELISA)and the data are depicted in FIG. 17, which shows TNFα concentrationsafter 3-hour cultures of 220 μg/ml (WBC alone), 160 μg/ml (WBC plusPeptideX2 at 10 ug/ml), 76 μg/ml (WBC plus PeptideX2 at 100 ug/ml), and0.05 μg/ml (serum plus PeptideX2 at 100 ug/ml). In this experiment, thepresence of PeptideX2 at 10 ug/ml resulted in 27% reduction in TNFαproduction at the 3-hour timepoint; when 100 ug/ml PeptideX2 was usedthere was a 65% reduction in TNFα production. In this three-hourtimeframe, however, the presence of PeptideX2 at these concentrationshad no effect on WBC bacteriocidal activity against S. aureus.

Example 14 In Vivo Sepsis Models

A. Cecal Ligation and Puncture Model. The observation that PeptideX2delivers a biological signal via specific recognition of murine TLR2and/or TLR4 is exploited in the cecal ligation and puncture (CLP)method, a recognized model for sepsis (Kasten et al., 2010 Infect.Immun. 78:4714).

C57BL/6 (WT) mice between 6 and 8 weeks of age (20-28 gms) are obtainedfrom Jackson Laboratory, Bar Harbor, Me.; all experiments involvinganimals are performed under protocols approved by the InstitutionalAnimal Care and Use Committee (IACUC). Polymicrobial sepsis is inducedusing the CLP method. Briefly, well fed mice are anesthetized to effectby 2.5% isoflurane in oxygen via face masks. After laparotomy, thelatter 80% of the cecum is ligated and punctured once on theanti-mesenteric side with a 23-gauge needle. A small amount of bowelcontent is extruded through the puncture hole to ensure full thicknessof the perforation. The cecum is replaced to its original location, andthe midline incision is closed by a two-layer suture.

Prior to closure of the peritoneum with one figure-of-eight stitch, 0,25, 125, 250, or 500 ug quantities of PeptideX2 [SEQ ID NO:2] (each in atotal volume 250 ul) are injected into the peritoneum or an equivalentvolume of saline so that the total concentration in such test animals is0, 10, 50, 100, or 200 ug/ml respectively. Sham-operated animals receivemidline laparotomies, exteriorization of the cecum with promptreplacement, and closure of incisions in two layers. The animals areresuscitated with 1 ml of sterile saline subcutaneously and kept on aheating blanket and additional oxygen supply for 1 hr. Mice receiving 0,10, 50, 100, or 200 ug/ml of PeptideX2 (total volume 250 ul) continue toreceive this same dosing as subcutaneous injections daily starting 24hours after the laparotomy and for which the total volume is the samefor all test mice. Sham-treated mice receive daily saline subcutaneousinjections of the same volume as used in the test mice starting 24 hoursafter the laparotomy. In survival studies, animals are given ad libitumaccess to food and water and followed until death or humane sacrificeper protocol. Animals are evaluated every 12 hr following CLP.

In separate test animals PeptideX2 is also administered at the variousdosing amounts as described above but at delayed time periods post cecalligation and puncture, using subcutaneous dosing every 24 hours todetermine if the product can be lifesaving in later stages of sepsis.Pharmacokinetics of PeptideX2 in vivo, quantification of totalcirculating white blood cells and of neutrophils and CD4+ and CD8+T-cell subsets, and quantification of cytokines, are undertakenaccording to standard methodologies.

B. Pneumococcal Pneumonia Model. Sepsis is often associated with severepneumonia, and pneumococcal PAMPs for TLR2 (lipoteichoic acid,lipopeptides) and TLR4 (pneumolysis) have been identified. Accordingly,an established murine pneumococcal pneumonia model is also used toobserve immunomodulatory effects of the herein described PeptideX2 andrelated immunomodulatory polypeptides, including effects of theseimmunomodulatory polypeptides that derive from their ability to competewith PAMPs as ligands for TLR2 and TLR4 and to deliver attenuated TLR2/4biological signaling. Briefly, Streptococcus pneumoniae loads (serotype3) are administered intratracheally to experimental animals before,during or after administration of PeptideX2 (control animals receive noPeptideX2; test groups receiving antibiotics alone or in addition toPeptideX2 are also contemplated). One or more biological indicia ofinfection, immunity, inflammation, and/or sepsis are determined atvarious timepoints, including animal survival, bacterial levels in theblood and lungs, lung histology, inflammatory mediators, appearance ofanti-bacterial antibodies, SIRS-like factors, and other criteria (e.g.,Chiavolini et al., 2008 Clin. Microbiol. Rev. 21(4):666-685; see also,e.g., Christaki et al., 2010 J. Infect. Dis. 201(8):1250; Christaki etal., 2011 Shock 35:492).

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent application, foreign patents, foreign patentapplication and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety.

Aspects of the embodiments can be modified, if necessary to employconcepts of the various patents, application and publications to provideyet further embodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

What is claimed is:
 1. An isolated polyclonal antibody, orantigen-binding fragment thereof, that specifically binds to animmunomodulatory polypeptide of no more than 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 amino acids, saidimmunomodulatory polypeptide comprising the amino acid sequence setforth in SEQ ID NO:2.
 2. A pharmaceutical composition comprising theantibody of claim 1 and a physiologically acceptable carrier.
 3. Amethod for detecting, in a biological sample, an that comprises theamino acid sequence set forth in SEQ ID NO:2, said method comprising thesteps of: (a) contacting the biological sample with the polyclonalantibody or antigen-binding fragment thereof of claim 1, underconditions and for a time sufficient for specific antibody binding tothe immunomodulatory polypeptide to take place; and (b) detectingspecific binding of the polyclonal antibody or antigen-binding fragmentthereof to the immunomodulatory peptide, and thereby detecting theimmunomodulatory peptide in the sample.
 4. The method of claim 3 whereinat least one of: (i) the polyclonal antibody or antigen-binding fragmentthereof is linked to a support material, (ii) the polyclonal antibody orantigen-binding fragment thereof is linked to a detectable label, or(iii) the biological sample is obtained from a subject that is selectedfrom a human, a non-human primate, a non-primate mammal, a non-mammalianvertebrate, an invertebrate eukaryote and a prokaryote.